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Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR.

ABSTRACT: A PCR assay was developed by using degenerate primers that allow amplification of a 414-bp fragment of DNA from the rickettsia-like organisms Rochalimaea henselae and R. quintana. Internal oligonucleotides were used as hybridization probes, permitting rapid differentiation of these two Rochalimaea species. DNAs from 12 different isolates of R. henselae were amplified with the PCR primers, and the resulting 414-bp PCR product hybridized only with the R. henselae-specific probe. DNAs from four different isolates of R. quintana were amplified and produced a PCR product of the same size that hybridized only with the R. quintana-specific probe. DNAs from isolates of R. elizabethae, R. vinsonii, Bartonella bacilliformis, and Afipia felis failed to amplify the 414-bp fragment in the PCR assay. This two-step assay was applied to DNAs extracted from 16 fresh (unfixed) lymph node biopsy specimens and nine aspirates from patients with clinical cat scratch disease (CSD) to assay for the presence of R. henselae or R. quintana DNA in these samples. Twenty-one (84%) of 25 lymph node samples from CSD patients were positive for R. henselae, while none were positive for R. quintana. The characteristic 414-bp fragment was not amplified from eight lymph node tissue samples from non-CSD cases. These results provide evidence that R. henselae, and not R. quintana, plays the central role in the etiology of CSD.

SUBMITTER: Anderson B

PROVIDER: S-EPMC263167 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

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Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR.

Anderson B B Sims K K Regnery R R Robinson L L Schmidt M J MJ Goral S S Hager C C Edwards K K

Journal of clinical microbiology 19940401 4

A PCR assay was developed by using degenerate primers that allow amplification of a 414-bp fragment of DNA from the rickettsia-like organisms Rochalimaea henselae and R. quintana. Internal oligonucleotides were used as hybridization probes, permitting rapid differentiation of these two Rochalimaea species. DNAs from 12 different isolates of R. henselae were amplified with the PCR primers, and the resulting 414-bp PCR product hybridized only with the R. henselae-specific probe. DNAs from four dif ...[more]

PMID: 8027347

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Project description:Cat scratch disease (CSD) is an infectious disease caused by Bartonella henselae, usually characterized by self-limiting regional lymphadenopathy and fever. Given the low clinical diagnostic sensitivity and specificity of conventional anti-B. henselae indirect immunofluorescence assays (IFAs), real-time polymerase chain reaction (PCR)-based detection of B. henselae is now being proposed as a more sensitive tool to diagnose CSD. Thus, here we have assessed the efficacy of real-time PCR in detecting B. henselae in different specimens from patients with suspected CSD and compared it to that of IFA. From March 2011 to May 2016, at the Microbiology and Virology Unit, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Turin, Italy, 115 clinical specimens (56 aspirated pus, 39 fresh lymph node biopsies, and 20 whole blood samples) and 99 sera from 115 patients with suspected CSD (62 females and 53 males between the ages of 3 months and 68 years) were analyzed by both real-time PCR, used in a qualitative way, and IFA (IgM and IgG) for the presence of B. henselae. For 16 patients, serological results were not available due to a clinical decision not to request the test. B. henselae DNA positivity was detected by real-time PCR in 37.39% of patients, while 62.61% of them were negative. Thus, patients were divided into two groups: real-time PCR+ (n = 43) and real-time PCR- (n = 72). Real-time PCR screening of whole blood, biopsies, and aspirated pus revealed B. henselae positivity in 40%, 38.46%, and 35.71% of patients, respectively. When we analyzed samples by IFA, we found the presence of B. henselae in 28 out of 99 (28.28%) patients, of which 11 (11.11%) belonged to the real-time PCR+ group and 17 (17.17%) to the real-time PCR- group. Among the 71 seronegative subjects, 16 (16.16%) were found positive for B. henselae by real-time PCR. Thus, by combining the results of both assays, we were able to increase the percentage of B. henselae positive specimens from 27.27% (real-time PCR) or 28.28% (IFA) to 44.44% (real-time PCR+IFA). Altogether, these findings indicate that the early detection of B. henselae in patients with suspicious CSD through combined real-time PCR and serological analyses can lead to a more accurate diagnosis of CSD, thereby allowing prompt and appropriate disease management.

Project description:Bartonella henselae is a Gram-negative bacterium that causes cat scratch disease (CSD), as well as bacteremia, endocarditis, and other clinical presentations. CSD remains one of the most common infections caused by bacteria in the genus Bartonella, and it is transmitted to humans through a scratch or cat bite. Vaccination and more efficient diagnostic methods would represent a promising and sustainable alternative measure for CSD control in humans and animals. Here, we described the in silico analyses and design of three recombinant chimeric proteins (rC1, rC2, and rC3), for use in the control of CSD. The chimeras were constructed with epitopes identified from the sequences of the GroEL, 17 kDa, P26, BadA, Pap31, OMP 89, and OMP 43, previously described as the most important B. henselae antigens. The rC1, rC2, and rC3 were expressed and purified using a heterologous system based on Escherichia coli and reacted with antibodies present in the sera of humans naturally infected. The chimeric proteins were used to immunize mice using Freund adjuvant, and the humoral immune response was evaluated. Animals immunized with rC1 and rC3 showed a significant IgG antibodies response from the 28th day (P < 0.05), and the animals immunized with the rC2 from the 35th day (P < 0.05) remained until the 56th day of experimentation, with a titer of 1:3200 (P < 0.05), 1:1600 (P < 0.05) and 1:1600 (P < 0.05) from rC1, rC2, and rC3, respectively. Significant production of IgA and IgG1 isotype was detected in animals immunized with rC1 and rC2 proteins. Additionally, analysis using 13 serum samples from naturally infected patients showed that the proteins are recognized by antibodies present in sera, reinforcing the possibility of using these chimeras for CSD control. KEY POINTS: • The recombinant chimeras were expressed in Escherichia coli with 37 kDa (rC1), 35 kDa (rC2), and 38 kDa (rC3). • Animals immunized with rC1, rC2, and rC3 showed significant antibody response. • The chimeras were recognized by the sera of naturally infected patients.

Dataset Information

Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR.

Publications

Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR.

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