Rapid polymerase chain reaction-based confirmation of cat scratch disease and Bartonella henselae infection.
BACKGROUND
Cat scratch disease (CSD) commonly occurs secondary to Bartonella henselae infection, and the diagnosis has traditionally been made by microscopic findings, the identification of organisms by cytochemistry, and clinical history. However, cytochemical analysis tends to be very difficult to interpret, and histology alone may be insufficient to establish a definitive diagnosis of CSD.


OBJECTIVE
To demonstrate the presence of B henselae in tissue suspected of involvement by CSD, using a novel polymerase chain reaction (PCR) assay.


METHODS
Isolates of B henselae (American Tissue Culture Collection 49793) and Afipia felis (American Tissue Culture Collection 49714) were cultured on blood agar and buffered charcoal yeast extract agar, respectively. DNA was isolated from these organisms and from formalin-fixed, paraffin-embedded tissue sections with involvement by CSD (8 patients). Negative controls included water, human placental tissue, and lymph node specimens from 6 patients with reactive lymphoid hyperplasia and from 2 patients with granulomatous lymphadenitis. A primer complementary to B henselae citrate synthase gltA gene sequence was designed to perform a seminested PCR amplification. For restriction fragment length polymorphism analysis, PCR products were digested by TaqI restriction enzyme and analyzed by gel electrophoresis.


RESULTS
Seminested PCR analysis of the cultured isolates of B henselae, but not of A felis, showed specific amplification. However, nonnested PCR did not provide consistently positive results in tissue sections with CSD. Therefore, we used a seminested PCR, which revealed positivity in all of the cases with clinicopathologic diagnoses of CSD. None of the negative controls showed positivity. Restriction enzyme provided confirmation of the specific PCR amplification of the B henselae sequence.


CONCLUSIONS
Since the amplification product has a low molecular size (<200 base pairs), this assay is useful for detection of B henselae in formalin-fixed, paraffin-embedded tissues. The seminested PCR protocol described here can be used for rapid and reliable confirmation of B henselae in samples that are histologically suggestive of CSD.
Department of Pathology, Loyola University Medical Center, Maywood, Ill 60153, USA.
Ben
B
Margolis
Isinsu
I
Kuzu
Marille
M
Herrmann
Michele D
MD
Raible
Eric
E
Hsi
Serhan
S
Alkan
D000293:Adolescent; D020622:Afipia / Q000235:genetics; D018416:Bartonella henselae / Q000235:genetics* / Q000302:isolation & purification*; D002372:Cat-Scratch Disease / Q000175:diagnosis*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005557:Formaldehyde / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D006801:Humans; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016612:Paraffin Embedding; D010920:Placenta / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011446:Prospective Studies; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012189:Retrospective Studies; D016707:Tissue Fixation
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D005557:Formaldehyde