Abstract
We evaluated the performance of tools for diagnosing Q fever cardiovascular infection. We retrospectively analyzed 162 cardiovascular samples from 125 patients who were tested serologically by immunofluorescence, quantitative PCR (qPCR), 16S rRNA gene amplification, culture, and immunohistochemistry, and we assessed the viability of Coxiella burnetii by measuring the transcription of the 16S rRNA gene. The qPCR technique was significantly more sensitive than 16S rRNA gene amplification (P < 0.0001), cell culture (P = 0.0002), and immunohistochemistry (P < 0.0001). The sensitivity of these techniques was reduced when applied to patients who had been previously treated. The severity of infection appears to be correlated with phase I IgG levels. We report for the first time 4 cases of endocarditis with positive qPCR and/or culture assay result from patients with a low phase I IgG (IgG I) titer (<800), and we have identified the longest (16 years) persistence of DNA described in a heart valve from a patient cured after being previously treated for endocarditis. The active transcription of the 16S rRNA gene was found in 19/59 tested samples, with a positive predictive value of 100% for a positive culture. In conclusion, the diagnosis of Q fever cardiovascular infection should not be excluded in patients with low titers of phase I IgG when they present with valvulopathy. We recommend testing cardiovascular samples using 3 or 4 different biopsy sections by qPCR evaluation for patients with IgG I titers of ≥200.
INTRODUCTION
Q fever is a ubiquitous zoonosis caused by infection with Coxiella burnetii, an obligate intracellular bacterium that can cause Q fever infections in humans (1). While most acute infections (60%) are asymptomatic, frequently observed symptoms include isolated fever, atypical pneumonia, and hepatitis (2, 3). From 1 to 5% of patients develop endocarditis or vascular infections, including infection in aneurysms or prostheses (4–6). The progression of infection occurs predominantly in patients with underlying cardiovascular abnormalities (5, 7). Serological analysis with an indirect immunofluorescence antibody (IFA) assay remains the most common method for diagnosing Q fever (8). Q fever cardiovascular infection can be diagnosed by isolating C. burnetii by using cell culture or by detecting C. burnetii using quantitative PCR (qPCR) or immunohistochemistry (9). Culture-based methods have a low sensitivity, require several weeks, and should be performed in a specialized biosafety level 3 laboratory. Alternatively, qPCR has been successfully used to detect C. burnetii DNA rapidly in various samples, such as serum, blood, cardiac valves, biopsy samples, and pharyngeal swabs (8). Treatment of Q fever endocarditis consists of administering doxycycline and hydroxychloroquine for 18 months in patients with native valves and 24 months in patients with prosthetic valves (10).
The objective of this study was to compare the performance of the diagnostic tools used on cardiovascular samples to diagnose Q fever cardiovascular infection. We developed a new tool to evaluate the viability of C. burnetii by measuring the transcription of the 16S rRNA gene in cardiovascular tissue (11).
MATERIALS AND METHODS
Sample collection.
As the French National Reference Center for Q fever, we receive samples from all regions of France, as well as other countries, for Q fever diagnosis. We included in the study all the patients for whom we received cardiovascular biopsy specimens between January 1999 and April 2013 who presented with definite and possible Q fever endocarditis or vascular infection, according to our updated criteria (8). A control group of 190 patients for which we excluded cardiovascular infection according to our recent criteria (8) during the last 14 years was used to evaluate the specificity of these techniques.
Cell culture.
The cardiac valves and vascular tissues were kept frozen at −80°C. All the samples from patients with a positive PCR and/or serology for C. burnetii were inoculated onto human embryonic lung fibroblasts growing in shell vials. Detection of C. burnetii growth within the cells was identified by Gimenez staining, immunofluorescence with in-house-prepared rabbit polyclonal antiserum, and qPCR, as previously described (12).
PCR assay.
DNA was extracted from surgically excised valvular or vascular tissue with a QIAamp tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Before 2004, specific PCR for C. burnetii was performed as described elsewhere (9). Since 2004, we have routinely used qPCR with the primers and TaqMan probes derived from the IS1111 repeated sequence (13) and using a LightCycler instrument (Roche Diagnostics GmbH, Germany). A standard calibration curve that provides quantification of the target was generated in a previous study by using 10-fold serial dilutions of C. burnetii (14). A standard internal control with a five-point 10-fold serial dilution of DNA of the C. burnetii Nine Mile II strain was used for each run of qPCR. We confirmed all the positive results with a qPCR targeting a second C. burnetii gene, the IS30a repeated sequence (14). For the samples obtained between 1999 and 2004, the DNA was kept frozen at −20°C and qPCR was performed retrospectively on these frozen samples.
The DNA from the Nine Mile II strain was used as a positive control, and sterile water was used as a negative control. The human actin gene was detected in parallel to verify the quality of the extracted DNA. A case was defined by 2 positive PCR results in assays targeting 2 different C. burnetii DNA sequences with threshold cycle (CT) values of ≤38.
The amplification of the 16S rRNA gene and the sequencing of the positive samples were performed as previously described (15).
Immunohistochemical analysis.
Immunohistochemistry was performed on paraffin-embedded valve sections with an anti-C. burnetii Cb10B10 mouse monoclonal antibody, as previously described (16).
Serology.
The serological tests were performed with an IFA assay, which is the reference method for the serodiagnosis of Q fever, as previously described (9).
Determination of MIC of doxycycline.
The MICs of doxycycline were determined by cell culture associated with qPCR, as previously described (17).
RT-qPCR.
Reverse transcriptase qPCR (RT-qPCR) was performed on 59 cardiovascular samples, including 46 valve tissues, 2 thrombi, 9 vascular samples, and 2 aortic prostheses kept frozen at −80°C. The sample disruption was performed with a TissueLyser II (Qiagen, Courtaboeuf, France) for 10 min at 25 Hz. The total RNA extraction and purification were performed with an RNeasy minikit (Qiagen, Courtaboeuf, France), and the DNase treatment was performed with Turbo DNase (Ambion, Courtaboeuf, France) according to the manufacturer's protocol. The total RNA profile size was evaluated with an Agilent 2100 Bioanalyzer instrument using the RNA 6000 Pico LabChip kit (Agilent Technologies, Massy, France). The one-step RT-qPCR (with SuperScript III platinum; Invitrogen) was performed on a Smart Cycler II (Cepheid, Maurens-Scopont, France) with primers and probes targeting the 16S rRNA of C. burnetii, CBU-16S-F, 5′-ACGGGTGAGTAATGCGTAGG-3′, CBU-16S-R, 5′-GCTGATCGTCCTCTCAGCC-3′, and CBU-16S-P, 6-carboxyfluorescein–GCAAAGCGGGGGATCTTCGG. The CT cutoff for positive results was 38.
Treatment data.
The data regarding treatment with doxycycline and hydroxychloroquine were acquired retrospectively for 100 patients, corresponding to 128 samples, by communicating with the physician responsible for each patient.
Statistical analyses.
For the data comparisons and statistical analyses, Student's t test, Fisher's exact test, or the χ2 test and standard statistical software (GraphPad Prism 5) were used. P values of <0.05 were considered significant.
RESULTS
Patient data.
Between January 1999 and April 2013, we tested 162 samples from 125 patients with Q fever cardiovascular infection, 104 with endocarditis, and 21 with vascular infection (Table 1). The mean age of the patients was 57 years (median, 59 years; range, 15 to 93 years), and 81% were male. A total of 54 patients were treated with doxycycline and hydroxychloroquine before surgery, with a mean duration of 16 months (median, 4 months; range, 1 to 36 months), and 46 patients had no previous antibiotic treatment.
Table 1.
Sources of samples received in our laboratory from January 1999 to April 2013a
| Patient type and source of sample | |
|---|---|
| Endocarditis (n = 104 patients) | Vascular infection (n = 21 patients) |
| 92 native cardiac valves | 7 thrombi |
| 5 valvular mitral prostheses | 12 vascular prostheses |
| 19 valvular aortic prostheses | 9 aortic tissue fragments |
| 1 pacemaker | 3 aneurysms |
| 1 bone marrowb | 6 aortic collections |
| 1 vein transplant | |
| 1 aortic fistula | |
| 1 aortic stent | |
| 2 retrosternal abscesses | |
| 2 paravertebral abscesses | |
A total of 162 samples were received from a total of 125 patients.
Bone marrow was sampled from patients with endocarditis according to updated criteria, and valvular samples were not received from this patient.
Sensitivities and specificities of PCR, culture, and immunohistochemistry.
Of the 162 samples tested, 127 were positive for C. burnetii by qPCR (78%), and 89 yielded positive cultures (55%) (Table 2). Immunohistochemistry was performed on 101 samples, of which 27 (27%) were positive (Table 2). 16sRNA amplification was performed on 60 samples, of which only 22 (37%) were positive for C. burnetii.
Table 2.
Results of culture, qPCR, and immunohistochemical analyses of the clinical samples
| Method of analysis for valvular and vascular surgical samples (n) | No. (%) with result |
|
|---|---|---|
| Positive | Negative | |
| 16S RNA gene amplification (60) | 22 (37) | 38 (63) |
| qPCR (162) | 127 (78) | 35 (22) |
| Shell vial culture (162) | 89 (55) | 73 (45) |
| Immunohistochemicistry (101) | 27 (27) | 74 (73) |
The results of the qPCR, culture, and immunohistochemical analysis were compared for 101 samples: 85% were positive by qPCR, 62% by culture, 27% by immunohistochemistry, and 22% by all 3 techniques. The results showed that qPCR was significantly more sensitive than cell culture and immunohistochemistry (P = 0.0002 and P < 0.0001, respectively, according to the χ2 test), and the specificities of these 3 techniques were 100%, as none of the negative control samples was positive.
We studied the effects of patient antibiotic treatment on the qPCR, culture, and immunohistochemistry results (Table 3). The sensitivities of qPCR (80%), culture (74%), and immunohistochemistry (35%) were higher for patients without treatment before surgery than for patients who received treatment, for whom sensitivity was 77%, 35%, and 18%, respectively. The detection of C. burnetii DNA was significantly more frequent for patients without treatment or with a treatment for less than 3 months than for patients treated for more than 3 months (P = 0.0035 using the χ2 test). Similarly, the means ± standard deviations (SD) of the CT values were significantly lower for patients without treatment (21.3 ± 6.6; P = 0.001) and for patients treated for less than 3 months (23.0 ± 5.3; P = 0.019) than for the patients treated for more than 3 months (27.6 ± 7.3), according to Student's t test (Fig. 1A). The percentage of positive culture and immunohistochemistry decreased in relation with the duration of antibiotic treatment. The number of positive cultures was significantly higher for patients who were not treated (P < 0.0001, using the χ2 test), but no significant difference among immunohistochemistry results was observed.
Table 3.
Comparison of methods for detecting C. burnetii in valves or vascular tissues from patients with Q fever endocarditis or vascular infection, according to the duration of therapy with doxycycline and hydroxychloroquine
| Detection method | No. (%) positive after: |
||
|---|---|---|---|
| No treatment | Treatment for ≤3 mo | Treatment for >3 mo | |
| qPCRa | 55/69 (80) | 29/31 (94) | 17/29 (59) |
| Cultureb | 51/69 (74) | 18/31 (58) | 3/29 (10) |
| Immunohistochemical analysis | 15/43 (35) | 4/18 (22) | 3/21 (14) |
DNA from C. burnetii was detected significantly more frequently in patients with less than 3 months of treatment or in those with no treatment before surgery than from patients treated for more than 3 months (P = 0.0035 using the χ2 test).
The number of positive cultures was significantly higher for patients who had not been treated with antibiotics (P < 0.0001 using the χ2 test).
Fig 1.
(A) The mean ± SD CT values obtained by qPCR for the cardiovascular biopsy specimens according to the duration of antibiotic treatment. Results were as follows: for cardiovascular biopsy specimens from patients who received no treatment, 21.3 ± 6.6 (●); for patients who received treatment for less than 3 months, 23.0 ± 5.3 (■); for patients who received treatment for more than 3 months, 27.6 ± 7.3 (▲). The positive cultures are indicated in red. (A) The relationship between CT values from qPCR and 16S rRNA gene amplification. The mean ± SD CT values obtained by qPCR for cardiovascular biopsy specimens according to 16S DNA gene amplification were as follows: positive, 18.4 ± 5.2 (●); negative, 25.7 ± 5.9 (■). (C) The relationship between the qPCR CT and 16S rRNA detection with RT-qPCR. The mean ± SD of the qPCR CT value for cardiovascular biopsy specimens in which 16S rRNA was detected was 19.7 ± 5.7 (●), and for cardiovascular biopsy specimens in which 16S rRNA was not detected, 24.5 ± 6.8 (■). The positive cultures are indicated in red.
Discrepancy between PCR, culture, and immunohistochemistry.
Multiple samples were received for 25 of the patients, and there was a discrepancy in the qPCR results between the different samples received in 5 cases and in the culture results of 6 cases. No discrepancies were noted between different samples derived from the same patient based on immunohistochemistry. No differences were observed between native valves and prosthetic samples with respect to the PCR, culture, or immunohistochemistry results.
The qPCR assay and culture were performed on 162 samples. Only one sample was positive by culture and negative by PCR, and 39 samples were positive by qPCR and negative by culture. Among these discrepant samples, the average length of treatment before surgery was 12 months, which is significantly higher than the average 1 month of treatment before surgery for the samples that were positive by qPCR and culture (P = 0.0001 using Student's t test). The discrepant results for untreated patients most likely resulted from performing the qPCR and culture on 2 different sections of the sample, because C. burnetii infection in valvular samples is restricted to a small area (16). We observed a positive correlation between the bacterial load estimated by qPCR and the success of isolation. The means ± SD of the qPCR CT values were significantly lower (21.9 ± 6.2) for the samples with positive cultures than for the samples with negative cultures (27.3 ± 6.5; P < 0.0001 using Student's t test).
16S rRNA gene amplifications with standard PCR and specific qPCR were performed on 60 samples, and qPCR (97%) was significantly more sensitive than the 16S rRNA gene amplification (37%; P < 0.0001 using the χ2 test). A positive 16S rRNA gene amplification was more significantly associated with a lower qPCR CT value (18.4 ± 5.2) than were samples that were negative for 16S rRNA gene amplification (25.7 ± 5.9; P < 0.0001 using Student's t test) (Fig. 1B).
Correlation between qPCR, culture, and serology results.
We compared the proportion of cardiovascular specimens that were positive for C. burnetii by qPCR and culture according to the phase I IgG (IgG I) titer in patients with less than 3 months of treatment (n = 57) (Table 4). The proportion of specimens that were positive by qPCR and/or culture increased significantly with elevated IgG phase I titer. In patients with an IgG I titer of ≤3,200, 59% were positive by qPCR and 50% by culture. This result is in contrast with those for patients with a titer of IgG I of ≥6,400, for whom 91% were positive by qPCR and 83% by culture (P = 0.0063 and P = 0.016, using Fisher's exact test). However, 4 patients without previous treatment presented cardiovascular infection with a positive qPCR and/or culture with an IgG I titer of <800; 3 patients had an IgG I titer of 400, and one had an IgG I titer of 200.
Table 4.
Proportion of positive cardiovascular specimens by qPCR and culture according to the phase I IgG titer in patients with less than 3 months of treatment (n = 57)
| Phase I IgG titer | No. of patients with cardiovascular infection | No. (%) positive by qPCR | No. (%) positive by culture | No (%) positive by both qPCR and culture |
|---|---|---|---|---|
| 200 | 1 | 1 (100) | 1 (100) | 1 (100) |
| 400 | 3 | 3 (100) | 2 (67) | 2 (67) |
| 800 | 12 | 7 (58) | 7 (58) | 7 (58) |
| 1,600 | 4 | 1 (25) | 0 (0) | 0 (0) |
| 3,200 | 2 | 1 (50) | 1 (50) | 1 (50) |
| 6,400 | 11 | 9 (82) | 9 (82) | 12 (82) |
| 12,800 | 6 | 6 (100) | 5 (83) | 7 (83) |
| 25,600 | 10 | 10 (100) | 9 (90) | 9 (90) |
| 51,200 | 4 | 4 (100) | 3 (75) | 4 (75) |
| 102,400 | 4 | 3 (75) | 3 (75) | 5 (75) |
Long-term persistence of DNA and viable bacteria.
DNA from C. burnetii was detected in valve tissue more than 18 months after the initiation of antibiotic treatment in 11 patients (Table 5). The maximum duration of DNA persistence in cardiac valves was 16 years, found for a 72-year-old man with Q fever endocarditis treated for 3 years and on whom cardiac surgery was performed 16 years later. Viable bacteria were detected in only one patient, a 59-year-old man with endocarditis that was treated for 19 months before surgery.
Table 5.
Long-term persistence of bacterial DNA and viable bacteria in patients who received 18 months of antibiotic treatment before surgerya
| Patient no. | Sample source | Result by detection method |
Duration of treatment before surgery (mo) | ||
|---|---|---|---|---|---|
| PCR | Culture | IHC | |||
| 1 | Cardiac valve | + | + | − | 19 |
| 2 | Cardiac valve | + | − | + | 20 |
| 3 | Cardiac valve | + | − | − | >36 |
| 4 | Cardiac valve | + | − | + | >36 |
| 5 | Cardiac valve | + | − | − | >36 |
| 6 | Cardiac valve | + | − | − | >36 |
| 7 | Cardiac valve | + | − | − | 30 |
| 8 | Cardiac valve | + | − | ND | 26 |
| 9 | Aortic stent | + | − | − | 30 |
| 10 | Cardiac valve | + | − | + | 24 |
| 11 | Cardiac valve | + | − | ND | 36 |
IHC, immunhistochemistry; ND, not determined.
MIC of doxycycline.
The doxycycline MIC was determined for the isolates from 68 patients, and it ranged from 0.25 to 8 μg/ml, with MICs of ≤1 μg/ml for 58 isolates and MICs of >1 μg/ml for 10 isolates. We did not observe an increase in the MIC of doxycycline over the past 14 years. No significant differences in the MIC were observed between the patients treated or not treated with antibiotics before cardiac surgery.
Bacterial viability.
We used RT-qPCR to determine bacterial viability by detecting the presence of C. burnetii 16S rRNA in cardiovascular tissue as proof of the transcriptional activity of the bacteria in these tissues. We used cell culture as our gold standard to verify bacterial viability and to compare our results. RT-qPCR targeting the 16S rRNA was performed retrospectively on 59 samples from 51 patients. All samples were positive by qPCR, and 43 (73%) were successfully cultured. C. burnetii 16S rRNA was detected in 19 samples (32%). All the samples containing C. burnetii 16S rRNA were associated with positive cultures, and 16S rRNA was not detected in any culture-negative samples. This test exhibited a 100% positive predictive value (PPV) for positive cultures. The 16S rRNA was not detected in 24 samples with positive cultures, and the negative predictive value was 40%. We studied the effect of antibiotic treatment on RNA detection from 40 patients, among which 24 were not treated and 16 were treated before surgery. C. burnetii 16S rRNA occurred more frequently in patients without treatment (12/24) than in patients treated before surgery (2/16; P = 0.015 using the χ2 test). It was established that the presence of 16S rRNA was more frequent in samples with a higher bacterial load (Fig. 1C). The mean ± SD CT values obtained by qPCR were significantly lower (19.7 ± 5.7) for the valvular samples containing 16S rRNA than for the valvular samples without detectable 16S rRNA (24.5 ± 6.8; P = 0.01 using Student's t test).
DISCUSSION
We have reported here the findings from the largest series of patients whose cardiovascular samples were used to perform microbiological diagnoses of Q fever cardiovascular infection. In our reference center, we tested these samples by qPCR, 16S rRNA gene amplification, culture, and immunohistochemistry, in addition to serology, to avoid false-positive and false-negative results and to increase the sensitivity for diagnosis. In our study, qPCR was positive in 78%, culture in 55%, immunohistochemistry in 27%, and 16S rRNA gene amplification in 37% of cases. Culture should be the most sensitive technique, with a detection limit of 1 living bacterium per sample, although qPCR detected a minimum of 30 to 40 bacteria per sample, including dormant or dead bacteria. Immunohistochemistry is known to be less sensitive, because C. burnetii can only be visualized within the region of inflammation in the macrophage cytoplasm, where the bacteria are numerous enough to be detected (Fig. 2) (16). Because the infective process may be confined, PCR and/or culture and/or immunohistochemistry may be negative (16). This limited area of infection may explain some of the observed dissonance between these three methods and between different samples from the same patient. The sensitivity of these techniques is related to the duration of antibiotic treatment before surgery (10, 16), particularly for culture- and immunohistochemistry-based analyses.
Fig 2.
Immunohistochemical detection of C. burnetii in the resected cardiac valve of a patient with Q fever endocarditis, showing that the infection may be very localized. A monoclonal antibody directed against C. burnetii and hematoxylin was used.
The qPCR technique is a useful, sensitive, specific, and rapid tool for the diagnosis of Q fever, compared to amplification of the 16S rRNA gene, even for samples with a low copy number of bacterial DNA (13, 18, 19). A recent report of C. burnetii prosthetic joint infection revealed that qPCR (and culture) is more sensitive than 16S rRNA gene amplification (20). Although qPCR with negative results was obtained from patients with endocarditis and high levels of phase I IgG, the negative findings do not definitively exclude the diagnosis of Q fever cardiovascular infection.
In previously treated patients, PCR remained positive but decreased significantly after 3 months of treatment in most cases, but DNA was able to persist for as long as 16 years postinfection in a patient apparently cured for 16 years. This persistence of DNA is the longest ever reported for bacterial endocarditis (21–24).
The extent of the infection appears to be correlated with the phase I IgG titer, and the proportion of positive qPCR results increases with elevated antibody titers. A previous study showed that an increase in the level of phase I IgG is correlated with higher positive predictive values for the diagnosis of endocarditis, and a threshold of ≥800 is typically retained (3). In our series, 4 patients presented definite cardiovascular infection with lower phase I IgG titers, including 3 patients with a titer of 400 and 1 with a titer of 200. We reevaluated the PPVs of phase I IgG titers for patient with a diagnosis of cardiovascular infection in an untreated group of valve replacement surgery patients; serology and qPCR were performed on the cardiovascular samples, and diagnoses were according to the updated criteria (8). The PPVs for the diagnosis of cardiovascular infection according to the IgG phase I titer were 53%, 68%, 71%, 79%, 88%, and 92% at titers of ≥200, ≥400, ≥800, ≥1,600, ≥3,200, and ≥6,400 for these patients (Table 6). In this context of a lower phase I IgG titer of ≤800, some endocarditis cases could not be confirmed without cardiovascular surgery. This work suggests that any patient with an IgG I titer of ≥200 should be tested by qPCR when the cardiac valves are removed.
Table 6.
PPV of phase I IgG titer for diagnosis of cardiovascular infection in an untreated group of valve replacement surgery patients
| Diagnosis | No. of patients with IgG I titer of: |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| ≥100 | ≥200 | ≥400 | ≥800 | ≥1,600 | ≥3,200 | ≥6,400 | ≥12,800 | ≥25,600 | ≥51,200 | ≥102,400 | |
| Definite endocarditis (n = 45) | 45 | 45 | 44 | 42 | 37 | 36 | 35 | 25 | 16 | 7 | 2 |
| All (n = 120) untreated valve replacement surgery patients (PPV [%]) | 120 (38) | 85 (53) | 65 (68) | 59 (71) | 47 (79) | 41 (88) | 38 (92) | 26 (96) | 17 (94) | 8 (88) | 3 (67) |
Culture is the gold standard for confirming bacterial viability, but C. burnetii is a demanding organism to culture. An interesting alternative strategy is to study the presence of bacterial RNA as a viability marker by quantifying the transcription of a housekeeping gene in clinical samples. A number of studies have shown that 16S rRNA disappears relatively rapidly after bactericidal treatment (25). In our study, the 16S rRNA of C. burnetii was used as a target and appeared to be a good marker of bacterial viability in cardiovascular samples. The 16S rRNA gene was detected significantly more frequently in patients who were not treated with antibiotics before surgery. An important limitation of our study is that RNA extraction and RT-qPCR were performed retrospectively on valve tissue kept frozen at −80°C and not treated with RNAlater reagent. If the negative predictive value of this test were low, it would exhibit a very good positive predictive value for a positive culture if 16S rRNA were detected in the sample. This innovative concept must be confirmed in a future study. Recently, RNA detection with RT-PCR has also been used to determine bacterial viability in other infectious diseases (26–29).
The diagnosis of Q fever cardiovascular infection should not be excluded in patients with low titers of phase I IgG when they present with valvulopathy. Consequently, we recommend using specific qPCR to test for the presence of C. burnetii in cardiovascular samples from patients with a predisposing heart condition who present with a phase I IgG titer of ≥200. Because the infection may be very localized, cardiovascular samples should be tested on 3 or 4 different biopsy sections to increase the sensitivity of the assay, particularly in a patient with a positive serology for which the first qPCR on cardiovascular samples was negative. The use of a new tool based on bacterial 16S rRNA detection to distinguish the presence of viable microorganisms appears to be an effective alternative to cell culture, which is technically and materially demanding. This finding remains to be confirmed.
ACKNOWLEDGMENTS
We thank Quentin Leroy, Karine Puggioni, Marie-Laure Birg, Jean-Yves Patrice, Annick Bernard, and Veronique Brice for their technical assistance.
All authors declare that they have no conflicts of interest.
This work was supported by the French National Referral Center for Q Fever.
Footnotes
Published ahead of print 12 July 2013
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