Diagnosis of Bartonella Endocarditis by a Real-Time Nested PCR Assay Using Serum

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_http://jcm.asm.org/cgi/content/full/41/3/919_

(http://jcm.asm.org/cgi/content/full/41/3/919)

 

Diagnosis of Bartonella Endocarditis by a Real-Time Nested PCR Assay Using

Serum

 

 

ABSTRACT

 

Bartonella endocarditis is a severe disease for which blood cultures

frequently remain negative. We tested three PCR assays by using specimens of

serum sampled early during the disease from 43 patients diagnosed in our

laboratory as having Bartonella endocarditis on the basis of serological,

culture, and/or valvular molecular detection. We tested a two-step nested PCR

(TSN-PCR), a one-step nested PCR (OSN-PCR) with a regular thermal cycler, and

a one-step nested PCR with the LightCycler (LCN-PCR). These assays were

performed with primers derived from the riboflavin synthase-encoding gene

ribC, never before amplified in our laboratory. Due to contamination of

negative controls, the results of the TSN-PCR were not interpretable, and this

technique was no longer considered. The LCN-PCR had a specificity of 100% and

a sensitivity of 58.1%, higher than those of the OSN-PCR (18.6%; P < 0.01)

and prolonged blood culturing (7.1%; P < 0.01). The LCN-PCR results

correlated strictly with those of other direct diagnostic tests, when

available,

and identified the causative species for six patients previously diagnosed

on the basis of serological analysis only. The efficacy of the LCN-PCR was

not influenced by antibiotics (P = 0.96) but was altered by prolonged

storage of serum specimens at -20°C (P = 0.04). Overall, the LCN-PCR is

specific

and more sensitive than traditional methods (i.e., culturing and/or PCR

with EDTA-treated blood). It can easily be applied to the diagnosis of

patients with suspected Bartonella endocarditis, especially when only serum is

available.

 

 

DISCUSSION

 

In the present report, we describe DNA detection methods for the diagnosis

of Bartonella endocarditis. Our purpose was to investigate the

effectiveness of PCR-based assays carried out on patients' serum specimens.

Serum is

one of the most easily obtained human samples and, when sampled early in the

evolution of a systemic disease, is likely to contain DNA copies of

systemic pathogens (28). Due to the presence of inhibitors in blood and to the

small amount of bacterial DNA present in serum, especially in patients

already treated with antibiotics, we designed three nested PCR assays to

increase

the sensitivity of PCR. A nested PCR approach was shown to be effective

for the diagnosis of rickettsial diseases and plague (26, 28, 30). A major

concern with nested PCR amplification is contamination, which may be lateral

(i.e., contamination caused by PCR products amplified in other tubes in the

same assay) or vertical (i.e., contamination caused by amplicons from

previous PCR assays).

To prevent vertical contamination, we used a suicide PCR assay

incorporating primers targeting a gene (ribC) never before amplified in our

laboratory

(2). In a first assay, we carried out a conventional nested PCR with two

successive amplifications in a standard thermal cycler. Unfortunately, in

this assay, all specimens, including negative controls, were positive.

Lateral contamination by aerosols containing amplicons from positive samples

probably occurred after the first PCR when the tubes were opened before the

second PCR. This result emphasizes the high risk of contamination of

conventional PCR assays.

 

To prevent such lateral contamination, we designed an OSN-PCR assay that

was performed in a different room. In this assay, the two primer pairs had

different hybridization temperatures and the reaction tubes were not opened

during the entire amplification process. Using one of the LightCycler

advantages, i.e., the ability to monitor the PCR cycle number before the onset

of amplification, we designed an LCN-PCR assay that enabled us to amplify

Bartonella DNA and detect contaminant amplification. We limited the number of

cycles in each of the two PCR assays to prevent the risk of nonspecific

amplification. Independently, a similar technique was recently reported for

the detection of RNA from hepatitis C virus (31). In parallel, we

investigated the usefulness of the technique with a standard thermal cycler

machine.

We observed no contamination of the negative controls during LCN-PCR or

OSN-PCR.

 

Overall, LCN-PCR exhibited a sensitivity of 58.1% and was more sensitive

than OSN-PCR. This result may be related to differences in PCR reagent

quality and enzyme stability and the short time needed to reach the annealing

temperature, which reduces the nonspecific annealing of primers. The

sensitivity of LCN-PCR was affected by the duration of serum storage at -20°C

(P =

0.04), likely through the progressive degradation of frozen DNA. In

addition to their longest storage times, the oldest serum specimens included in

our study had been thawed and refrozen repeatedly for various tests performed

in our laboratory, including MIF, cross-absorption, and Western blotting.

Repeated thawing of frozen samples may have played a role in the observed

decrease in sensitivity, as previously documented (9). In contrast, LCN-PCR

was not influenced by the administration of antibiotics. LCN-PCR was more

sensitive than blood culturing and valve sample culturing. However, LCN-PCR

performed on serum was less sensitive (19 of 26 patients) than PCR

performed on valve samples (25 of 26). The fact that the serum samples obtained

from six patients whose valve samples were found positive only by PCR had been

stored for more than 3 years may partially explain this difference in

sensitivity. Using OSN-PCR, we amplified Bartonella species from samples from

eight patients, including two for whom no direct evidence of a causative

agent had been obtained by other techniques. LCN-PCR detected Bartonella

species in samples from 25 patients. For 6 of these 25 patients, the infecting

species had not been identified by other direct diagnostic tests,

demonstrating the utility of this technique.

 

The ribC-derived LCN-PCR, with a one-step procedure in the LightCycler

thermal cycler, prevents amplicon carryover. We have demonstrated that this

technique can be carried out on serum specimens, especially before valvular

surgery, and is not affected by antimicrobial treatment. Its sensitivity is

higher when performed on serum samples kept frozen for short periods. In

conclusion, LCN-PCR is a valuable tool that may shorten the delay in the

diagnosis of Bartonella endocarditis. As Bartonella endocarditis represents 3%

of cases of infective endocarditis, we propose that this technique should

be applied to patients with blood culture-negative endocarditis and those

with both unexplained fever and elevated titers of antibodies to Bartonella

species. Moreover, this technique may be useful for other systemic

Bartonella infections, in particular, chronic bacteremia, bacillary

angiomatosis,

peliosis hepatis, and cat scratch disease with visceral involvement