|Year : 2015 | Volume
| Issue : 1 | Page : 185-186
16S rRNA PCR for the diagnosis of culture-negative Bartonella quintana endocarditis: The importance of sample type
YT Chin1, R Hasan2, A Qamruddin1
1 Department of Microbiology, Manchester Royal Infirmary, Manchester, United Kingdom
2 Cardiac Surgery Unit, Manchester Royal Infirmary, Manchester, United Kingdom
|Date of Submission||22-Jul-2013|
|Date of Acceptance||15-Aug-2013|
|Date of Web Publication||5-Jan-2015|
Department of Microbiology, Manchester Royal Infirmary, Manchester
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Chin Y T, Hasan R, Qamruddin A. 16S rRNA PCR for the diagnosis of culture-negative Bartonella quintana endocarditis: The importance of sample type. Indian J Med Microbiol 2015;33:185-6
|How to cite this URL:|
Chin Y T, Hasan R, Qamruddin A. 16S rRNA PCR for the diagnosis of culture-negative Bartonella quintana endocarditis: The importance of sample type. Indian J Med Microbiol [serial online] 2015 [cited 2020 Jul 6];33:185-6. Available from: http://www.ijmm.org/text.asp?2015/33/1/185/148429
Broad-range PCR for the detection of bacteria in clinical samples from normally sterile sites can be a useful diagnostic tool. In England, this service is provided by Public Health England (Colindale, United Kingdom) using a real-time polymerase chain reaction (PCR) for the amplification and sequencing of fragments within the universal bacterial ribosomal RNA gene (16S rRNA) from samples as diverse as blood, bone, CSF and tissue specimens.  Following a recent case we would like to emphasise that careful consideration should be given to the specimen to be submitted for broad-range PCR, depending on the clinical syndrome and possible pathogens.
The patient was admitted with a 4-month history of progressive shortness of breath, weight loss and fevers. A pan-systolic murmur was found on examination. X-ray computed tomography of the chest showed evidence of left upper lobe consolidation, with cardiomegaly and bilateral pleural effusions in keeping with severe mitral valve regurgitation [Figure 1]. Mitral regurgitation was confirmed on cardiac echocardiogram with vegetations seen on the mitral and pulmonary valves [Figure 2]. Despite four standard sets of blood cultures (BacTalert, Biomerieux), no organisms were isolated and the patient was commenced empirically on intravenous amoxicillin 2g q4h and gentamicin 1 mg/kg q12h for subacute bacterial endocarditis as per British Society of Antimicrobial Chemotherapy guidelines.  The patient deteriorated and proceeded emergently to surgery, where evidence of endocarditis was seen on the mitral valve with vegetations on the free edge of the valve and multiple chordae rupture. The prolapsing segment and vegetation were excised and an annuloplasty ring inserted. The vegetation seen on the right arterial leaflet of the pulmonary valve was excised without disrupting the valve leaflet. The excised mitral valve tissue and the pulmonary valve vegetation were submitted for culture.
|Figure 1: X-ray computed tomography of chest showing consolidation in the apicoposterior segment of the left upper lobe with adjacent architectural distortion and pulmonary nodules. There was also cardiomegaly and bilateral pleural effusions consistent with the history of severe mitral regurgitation|
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|Figure 2: Trans-thoracic echocardiography demonstrating evidence of (a) mitral valve prolapse; (b) mitral valve vegetation; and (c) pulmonary valve vegetation|
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At this time, results of serology were suggestive of bartonellosis and oral doxycycline 100 mg q12h was added. Cultures for all sample remained negative and, as culture for Bartonella sp. requires very specific requirements not available in our laboratory, the pulmonary valve vegetation, homogenised mitral valve tissue and peripheral blood EDTA sample (collected on admission, before starting antibiotics) were submitted for broad-range PCR to confirm the diagnosis. No DNA product was detected from the EDTA blood sample, but Bartonella quintana was identified from the mitral valve tissue (cycle threshold 24) and from the pulmonary valve vegetation (cycle threshold 14). The patient successfully completed 6 weeks of doxycycline with gentamicin for the initial 2 weeks and there were no further infective concerns at 4 months follow-up.
16S PCR has proven effective in the setting of blood culture-negative endocarditis  but most studies do not differentiate the sensitivity of broad-range PCR based on the sample type tested, such as between surgically removed valves, vegetations or blood samples. A literature search found only one study that did so, with broad-range PCR on EDTA blood providing a diagnosis in only 13.6% (35/257 patients) of cases, compared to 66.1% (150/227 patients) from valvular specimens in patients with blood culture-negative endocarditis.  Specifically for Bartonella endocarditis, this study found that broad-range PCR identified this organism from EDTA blood in only 12 patients compared with 26 from excised heart valve tissue.  This suggests that the sensitivity of 16S PCR is higher on valve tissue than for EDTA blood.
The difference in sensitivity of broad-range PCR between blood and tissue specimens may be explained by the use of antibiotics prior to sampling which may reduce the amount of bacterial DNA in peripheral blood, or the life cycle of the organism (Bartonella is a facultatively intracellular organism  so the amount of bacterial DNA may be lower in peripheral blood samples compared to tissue). Certain blood samples may not be ideal for 16S PCR; for example, sodium polyanetholesulfonate in blood culture fluid can inhibit the PCR amplification  and hence would be a poor sample for testing.
In addition to the differences between blood and tissue samples, our case also illustrated that the type of tissue sample may also influence the yield of broad-spectrum PCR, with 16S real-time PCR cycle thresholds of 14 from the vegetation and 24 from the valve tissue. The cycle threshold can be regarded as a semi-quantitative measurement of bacterial DNA, a value of <20 cycles indicative of a large amount of bacterial DNA and a value of >27 indicating a very low level.  This implies that the type of tissue biopsied may be critical, with vegetations more likely than heart valves to yield a positive result. This may not be surprising as vegetations are believed to contain dense clumps of bacteria (and hence high bacterial DNA loads) within a matrix of fibrin, platelets and erythrocyte debris. 
Molecular techniques such as broad-range PCR improve the likelihood of identifying an infectious agent but should be used judiciously and in conjunction with traditional diagnostics such as echocardiography, culture, serology and histology. Knowing the limitations of broad-range PCR from various samples is potentially important; for example, when it is not possible or appropriate to obtain valve tissue samples, only peripheral blood samples may be available for microbiological analysis. The importance of appropriate sample selection from normally sterile sites needs to be emphasised with selection of specimens that are most likely to have a significant bacterial DNA load.
| ~ References|| |
Edwards KJ, Logan JM, Langham S, Swift C, Gharbia SE. Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples. J Med Microbiol 2012;61:645-52.
Gould FK, Denning DW, Elliott TS, Foweraker J, Perry JD, Prendergast BD, et al
. Working Party of the British Society for Antimicrobial Chemotherapy. Guidelines for the diagnosis and antibiotic treatment of endocarditis in adults: A report of the Working Party of the British Society of Antimicrobial Chemotherapy. J Anticrob Chemother 2012;67:269-89.
Millar BC, Moore JE. Current trends in the molecular diagnosis of infective endocarditis. Eur J Clin Microbiol Infect Dis 2004;23:353-65.
Fournier PE, Thuny F, Richet H, Lepidi H, Casalta JP, Arzouni JP, et al
. Comprehensive diagnostic strategy for blood culture-negative endocarditis: A prospective study of 819 new cases. Clin Infect Dis 2010;51:131-40.
Rolain JM, Brouqui P, Koehler JE, Maguina C, Dolan MJ, Raoult D. Recommendations for treatment of human infections caused by Bartonella
species. Antimicrob Agents Chemother 2004;48:1921-33.
Fredricks DN, Relman DA. Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J Clin Microbiol 1998;36:2810-6.
Lepidi H, Casalta JP, Fournier PE, Habib G, Collart F, Raoult D. Quantitative histological examination of bioprosthetic heart valves. Clin Infect Dis 2006;42:590-6.
[Figure 1], [Figure 2]