Isolated hepatic involvement of cat scratch disease in immunocompetent adults: Enhanced magnetic resonance imaging, pathological findings, and molecular analysis--two cases

Visceral involvement in absence of lymphadenopathy is a rare manifestation in cat scratch disease; hepatic granulomas are rare, representing 0.3% of systemic manifestations of cat scratch disease, and gallbladder extension is a singular case. The present article refers to 2 rare cases of visceral cat scratch disease in immunocompetent adults with hepatic granulomatous inflammation, caused by Bartonella henselae infection, with gallbladder involvement in 1 case and no lymphadenopathy. Histological features demonstrated the presence of inflammatory necrotizing granulomatous nonneoplastic process. Molecular studies (polymerase chain reaction) were performed to confirm the infectious etiology.     

Isolation of Bartonella henselae DNA from the peripheral blood of a patient with cat scratch disease up to 4 months after the cat scratch injury

We report the case of a girl with cervical lymphadenitis and a persistent primary lesion of cat scratch disease (CSD). Bartonella henselae DNA was isolated from plasma samples collected 3 and 4 months after the cat scratch, indicating that recurrent and long-term shedding of Bartonella DNA into peripheral blood may occur in typical CSD.     

Cat scratch disease: detection of Bartonella henselae DNA in archival biopsies from patients with clinically, serologically, and histologically defined disease.

Serological and epidemiological studies suggest that Bartonella henselae is the etiological agent of cat scratch disease. We designed a study to detect B. henselae in archival biopsies by polymerase chain reaction amplification of the 16S rRNA gene followed by Southern blot hybridization. Forty-two histologically defined cat scratch disease biopsies and eighteen controls were selected for blinded analysis. After testing, charts were reviewed for clinical, immunological, and microbial evidence of infection. Results were correlated with duration of illness and antimicrobial therapy. B. henselae DNA was identified in 27 of 42 (64%) histologically defined patients and 23 of 34 (68%) patients defined both clinically and histologically. There were no false positives (0 of 18). A small subset (n = 14) had cat scratch disease serological tests performed. B. henselae was identified in 8 of 10 serologically positive patients. Polymerase chain reaction detected 50% of our DNA-positive cases (most of these early in the clinical course). Southern blotting of amplicons both doubled sensitivity (detecting patients > 4 weeks into illness) and confirmed B. henselae as the causative species. Our study strongly associates B. henselae with cat scratch disease, suggesting that it may be the most likely etiological agent in the majority of patients with cat scratch disease.     

Molecular diagnosis of cat scratch disease: a two-step approach.

Amplification of Bartonella henselae DNA has been proposed as a diagnostic test for cat scratch disease (CSD). The sensitivities of the following three PCR assays were compared. PCR/rRNA with universal primers amplifies part of the 16S rRNA gene, followed by hybridization with a specific B. henselae probe; PCR/CS and PCR/HSP amplify portions of the gltA and the htrA genes, respectively, each followed by restriction fragment length polymorphism analysis. The threshold of detection of B. henselae DNA in pus was 10(-4), 10(-3), and 10(-2) ng for PCR/rRNA, PCR/CS, and PCR/HSP, respectively. By these three assays, B. henselae DNA was detected in 100, 94, and 69% of 32 pus and lymph node specimens from CSD patients, respectively. The similar sensitivities of the PCR/rRNA and the PCR/CS assays for detecting B. henselae DNA in clinical specimens are in contrast to the 10-fold difference in sensitivities in favor of PCR/rRNA demonstrated with purified B. henselae DNA in sterile pus, suggesting that in the majority of cases, the bacterial load in clinical specimens is large enough to be identified by the PCR/CS assay. A two-step approach is suggested to achieve maximal sensitivity for detecting B. henselae in clinical specimens: initial testing by PCR/CS (which does not require hybridization), followed by PCR/rRNA with PCR/CS-negative specimens when CSD is strongly suspected.     

Cat scratch disease presenting as orbital abscess and osteomyelitis

Ocular manifestations of cat scratch disease are uncommon. The diagnosis is usually made on the basis of increasing Bartonella henselae serum antibody titers. We report a child presenting with orbital abscess and osteomyelitis who was diagnosed with hepatosplenic cat scratch disease by detection of B. henselae DNA in the orbital abscess fluid.     

Combining cytomorphology and serology for the diagnosis of cat scratch disease

Cat scratch disease (CSD) is a self limited zoonotic disease that presents most commonly as a regional lymphadenopathy. We are reporting a case of a 25-year-old male patient who presented with fever and large right inguinal lymphadenopathy. The diagnosis of cat scratch disease was confirmed based on the characteristic cytopathological features on aspirate smears from the lymph node and the serological titers for Bartonella henselae. This case report emphasizes the importance of combining Bartonella serology, and cytopathology in the diagnostic work-up of febrile lymphadenopathy and suspected CSD since the culture of this organism is arduous.     

Cervical cat scratch disease lymphadenitis in a patient with immunoglobulin M antibodies to Toxoplasma gondii

We report on a young patient with chronic cervical lymphadenopathy and serological and histological evidence for infection with Bartonella henselae and Toxoplasma gondii. Serological follow-up studies, including testing for avidity of Toxoplasma-specific immunoglobulin G antibodies, assisted in the determination of the cause of the acute lymphadenitis. Our results suggest that the clinical symptoms were most likely due to cat scratch disease rather than to acute toxoplasmosis.     

Experimental transmission of Bartonella henselae by the cat flea.

Bartonella henselae is an emerging bacterial pathogen, causing cat scratch disease and bacillary angiomatosis. Cats bacteremic with B. henselae constitute a large reservoir from which humans become infected. Prevention of human infection depends on elucidation of the natural history and means of feline infection. We studied 47 cattery cats in a private home for 12 months to determine the longitudinal prevalence of B. henselae bacteremia, the prevalence of B. henselae in the fleas infesting these cats, and whether B. henselae is transmitted experimentally to cats via fleas. Vector-mediated transmission of B.henselae isolates was evaluated by removing fleas from the naturally bacteremic, flea-infested cattery cats and transferring these fleas to specific-pathogen-free (SPF) kittens housed in a controlled, arthropod-free University Animal Facility. B. henselae bacteremia was detected in 89% of the 47 naturally infected cattery cats. A total of 132 fleas were removed from cats whose blood was simultaneously cultured during different seasons and were tested individually for the presence of B. henselae DNA by PCR. B. henselae DNA was detected in 34% of 132 fleas, with seasonal variation, but without an association between the presence or the level of bacteremia in the corresponding cat. Cat fleas removed from bacteremic cattery cats transmitted B. henselae to five SPF kittens in two separate experiments; however, control SPF kittens housed with highly bacteremic kittens in the absence of fleas did not become infected. These data demonstrate that the cat flea readily transmits B. henselae to cats. Control of feline infestation with this arthropod vector may provide an important strategy for the prevention of infection of both humans and cats.     

The diagnosis of cat-scratch-disease-associated adenitis: diagnostic value of serology and polymerase chain reaction

The diagnosis of cat scratch disease (CSD) associated adenitis relies classically on the association of clinical, epidemiological and bacteriological criteria. The polymerase chain reaction (PCR) looks like a more competitive diagnostic trial than serology. We evaluated the sensitivity, specificity and predictive positive and negative values of serology in routine diagnosis of CSD. A retrospective study over five years was led among patients presenting a suspicion of CSD and having a serology and/or a PCR. The Gold standard for diagnosis was PCR. The serological tests of Bartonella henselae was performed once in 482 patients, of which 2% (11 out of 482) were positive, and twice in only 39 patients (8%). The PCR diagnosis method for B. henselae was performed in biopsy of specimen lymph nodes in 28 patients and 14 out of 28 were positive. In nine patients, the diagnosis was exclusively made by PCR. Among the 14 patients whose PCR was negative, two had a positive serology and in three others patients, the serology was not performed. The sensitivity of serology was 35%, this confirms the low sensitivity of the serology in the CSD diagnosis. The diagnosis was confirmed in 56% of cases where PCR was performed. This led us to propose to perform systematically the PCR test for B. henselae in case of adenitis possibly associated with CSD.     

Lymphadenopathy in a novel mouse model of Bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/beta

In immunocompetent humans, cat scratch disease (CSD) is elicited by the Gram-negative bacterium Bartonella henselae and is characterized by a benign regional lymphadenopathy, the pathogenesis of which is poorly understood. Here, we describe a novel mouse model of Bartonella-induced CSD-like disease that allowed us to investigate the mechanisms leading to lymphadenopathy in vivo. In wild-type mice, a subcutaneous inoculation of either viable or inactivated B. henselae led to a strong swelling of the draining lymph node, which was long-lasting despite the rapid elimination of the bacteria. Carboxyfluorescein- and bromodesoxyuridine-labeling experiments showed that lymph node enlargement resulted from modified immigration and enhanced proliferation of lymphocytes, preferentially of B cells. A comparative analysis of B. henselae and the rodent pathogen B. grahamii in wild-type versus interferon-alpha/beta-receptor I chain-deficient mice revealed that interferon-alpha/beta is not only differentially induced by these two Bartonella species but also exerts an inhibitory effect on the development of lymphadenopathy both in vitro and in vivo. These data demonstrate that the lymphadenopathy of human CSD can be reproduced and studied in a mouse model and provide the first insights into the underlying immunological mechanisms.     

Evaluation of a in house serology reagent for the diagnosis of cat-scratch disease defined by PCR

AIM OF THE STUDY: evaluate the sensitivity and the specificity of the cat scratch disease serology by indirect immunofluorescence assay, realized from an in-house antigenic suspension, with PCR defined cases. Describe the epidemiological characteristics of the cases. METHODS: the antigenic suspension is realized by culture of a Houston 1 ATCC 49882 B. henselae reference strain on horse blood agar suspended in egg formoled PBS. Real time PCR from clinical samples is performed by amplification of a 998-bp 16S rDNA sequence with Bart and r-BH primers. RESULTS: In 57 out of 92 (62%) positive patients in PCR, the serology is positive in IgG at low or significative level or positive in IgG with presence of IgM or shows a seroconversion. The specificity in serum samples from 40 control patients is 100%. The average age of the 165 positive patients in PCR is 27.6 years old (3-80). The localization of the lymph nodes is more often axillary (47%) than inguinal (32%) or cervical (16%). CONCLUSION: Our in-house indirect immunofluorescence assay for the cat scratch disease serology shows a sensitivity equivalent to other technics described in the literature, with an excellent specificity.     

Low prevalence of Bartonella henselae infections in Norwegian domestic and feral cats

Bartonella henselae is the causative agent of cat scratch disease (CSD). This clinical entity is very rarely encountered in human medical practice in Norway. B. henselae infections including bacteraemia in cats have been frequently reported. The objective of the present study was to investigate the seroprevalence rate and the degree of B. henselae bacteraemia in Norwegian domestic and feral cats. One hundred cats investigated at a small animal veterinary practice in the middle of Norway were included in the study. Blood collected in Isolator blood-lysis tubes and lysates of erythrocytes after freezing and thawing were cultured. PCR analysis of whole blood was also performed. Serology was performed by indirect fluorescence assay (IFA) and enzyme immunoassay (EIA) using immobilised B. henselae Houston-1 strain as antigen. None of the 100 cats investigated was found to be bacteraemic. All 100 cats were seronegative when analysed by IFA; one cat was positive by EIA. The discrepancy between IFA and EIA of this particular cat is probably due to cross-reactive antibodies. Contrary to findings reported from several geographic regions, B. henselae infections in Norwegian cats appear to be virtually absent. This in turn may explain why CSD has not been reported in human medical practice in Norway.     

Identification of Bartonella species directly in clinical specimens by PCR-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment

It is now established that two species of Bartonella, namely, Bartonella henselae and B. quintana, cause bacillary angiomatosis in human immunodeficiency virus-infected patients. In addition, B. henselae causes cat scratch disease and B. quintana, B. henselae, and B. elizabethae can cause bacteremia and endocarditis in immunocompetent persons. We have developed a PCR-restriction fragment length polymorphism-based assay for direct detection and identification to species level of Bartonella in clinical specimens. This is accomplished by PCR amplification of Bartonella DNA using primers derived from conserved regions of the gene carrying the 16S ribosomal DNA, followed by restriction analysis using DdeI and MseI restriction endonucleases. We amplified a Bartonella genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from digestion of B. henselae and B. quintana amplicons with DdeI, whereas a different unique pattern was seen by using the same enzyme with B. vinsonii and B. elizabethae. With MseI digestion, B. henselae and B. vinsonii gave nearly identical patterns while B. quintana and B. elizabethae gave a different pattern. By combining the restriction analysis data generated with MseI and DdeI, unique "signature" restriction patterns characteristic for each species were obtained. These patterns were useful in identifying the Bartonella species associated with each tissue specimen.     

Molecular detection of Bartonella spp. in the dental pulp of stray cats buried for a year

Bartonella henselae causes chronic bacteremia in cats. To test if B. henselae DNA can be recovered from the dental pulp of cats buried a year previously, we used PCR with primers for a sequence of the conserved groEL to test 104 teeth from 11 cats. Seven of the cats were found positive; canine teeth were more frequently positive than molar teeth. Where PCR sequences could be determined, they were identical to those of B. henselae Marseille (four cats), B. henselae Houston (one cat) or similar to those of B. grahamii (one cat). Our study indicates that dental pulp from the teeth of cats, especially the canine teeth, may be used for the PCR detection of Bartonella in animals buried for a year.     

Bartonella clarridgeiae, a newly recognized zoonotic pathogen causing inoculation papules, fever, and lymphadenopathy (cat scratch disease).

Shortly after adopting a 6-week-old cat, a veterinarian was bitten on the left index finger. Within 3 weeks, he developed headache, fever, and left axillary lymphadenopathy. Initial blood cultures from the cat and veterinarian were sterile. Repeat cultures from the cat grew Bartonella-like organisms with lophotrichous flagella. Sera from the veterinarian were not reactive against Bartonella henselae, B. quintana, or B. elizabethae antigens but were seroreactive (reciprocal titer, 1,024) against the feline isolate. Sequential serum samples from the cat were reactive against antigens of B. henselae (titer, 1,024), B. quintana (titer, 128), and the feline isolate (titer, 2,048). Phenotypic and genotypic characterization of this and six additional feline isolates, including microscopic evaluation, biochemical analysis, 16S rRNA gene sequencing, DNA-DNA hybridization, and PCR-restriction fragment length polymorphism of the 16S gene, 16S-23S intergenic spacer region, and citrate synthase gene identified the isolates as B. clarridgeiae. This is the first report of cat scratch disease associated with B. clarridgeiae.     

Intracellular location of Bartonella henselae cocultivated with Vero cells and used for an indirect fluorescent-antibody test.

Bartonella henselae, the major causative agent of cat scratch disease, was cocultivated with Vero cells on chamber slides and visualized by indirect immunofluorescence by using a patient serum containing specific antibodies. Confocal microscopy localized the granular B. henselae-specific fluorescence mainly around the nuclei of Vero cells. By transmission electron microscopy, these granules were identified as clusters of multiple intracellular organisms. Fixed slides with the monolayers of Vero cells with intracellular B. henselae were used for an indirect fluorescent-antibody test to investigate the seroprevalence of specific immunoglobulin G in 100 serum samples from blood donors. Seventy-four serum samples were negative; 19, 3, and 4 were positive at dilutions of 1:64, 1:128, and 1:256, respectively. In our population, a serum titer of 1:256 or greater should stimulate further investigations. Moreover, elucidation of the mechanism by which B. henselae enters the cells may help to understand the pathogenesis of cat scratch disease.     

Study of genotypes and virB4 secretion gene of Bartonella henselae strains from patients with clinically defined cat scratch disease

Bartonella henselae is the causative agent of cat scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. Occasionally, the bacteria will spread and be responsible for tissue and visceral involvement. Two B. henselae genotypes (genotypes I and II) have been described to be responsible for uncomplicated CSD on the basis of 16S rRNA sequence analysis. A type IV secretion system (T4SS) similar to the virulence-associated VirB system of Agrobacterium tumefaciens was recently identified in the B. henselae Houston-1 genotype I strain. We studied the correlations of the B. henselae genotypes with the clinical presentations and with the presence of T4SS. Isolates originated from CSD patients whose lymph nodes were prospectively analyzed. B. henselae genotype I was identified in 13 of 42 patients (30%). Among these, two teenage twins presented with hepatosplenic CSD and one immunocompetent adult presented with osteomyelitis. Genotype II was detected in 28 of 42 patients (67%), all of whom presented with uncomplicated CSD. The last patient was infected with both genotypes. T4SS was studied by PCR amplification of the virB4 gene. Amplification of virB4 codons 146 to 256, 273 to 357, and 480 to 537 enabled us to detect 66, 90, and 100% of the B. henselae isolates, respectively. Sequence analysis revealed sequence variations that correlated with genotype distribution. Our studies suggest that B. henselae genotype I strains harbor virB4 genes that are different from those harbored by genotype II strains and that genotype I strains might be more pathogenic.     

Inter- and intraspecies identification of Bartonella (Rochalimaea) species.

Species of the genus Rochalimaea, recently renamed Bartonella, are of a growing medical interest. Bartonella quintana was reported as the cause of trench fever, endocarditis, and bacillary angiomatosis. B. henselae has been implicated in symptoms and infections of human immunodeficiency virus-infected patients, such as fever, endocarditis, and bacillary angiomatosis, and is involved in the etiology of cat scratch disease. Such a wide spectrum of infections makes it necessary to obtain an intraspecies identification tool in order to perform epidemiological studies. B. vinsonii, B. elizabethae, seven isolates of B. quintana, and four isolates of B. henselae were studied by pulsed-field gel electrophoresis (PFGE) after restriction with the infrequently cutting endonucleases NotI, EagI, and SmaI. Specific profiles were obtained for each of the four Bartonella species. Comparison of genomic fingerprints of isolates of the same species showed polymorphism in DNA restriction patterns, and a specific profile was obtained for each isolate. A phylogenetic analysis of the B. quintana isolates was obtained by using the Dice coefficient, UPGMA (unweighted pair-group method of arithmetic averages), and Package Philip programming. Amplification by PCR and subsequent sequencing using an automated laser fluorescent DNA sequencer (Pharmacia) was performed on the intergenic spacer region (ITS) between the 16 and 23S rRNA genes. It was found that each B. henselae isolate had a specific sequence, while the B. quintana isolates fell into only two groups. When endonuclease restriction analysis of the ITS PCR product was done, three enzymes, TaqI, HindIII, and HaeIII, allowed species identification of Bartonella spp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conservation of the 17-kilodalton antigen gene within the genus Bartonella

The 17-kDa antigen of Bartonella henselae has previously been shown to elicit a strong humoral immune response in patients with cat scratch disease (CSD) and to be useful in screening human serum samples for CSD. In this study, PCR amplification of genes homologous to the 17-kDa antigen gene of B. henselae was performed using genomic DNAs from several species of Bartonella, including the currently recognized human pathogens. Amplicons of similar size were demonstrated using the following chromosomal DNA templates: B. henselae (two strains), B. quintana (two strains), B. elizabethae, B. clarridgeiae, B. vinsonii subsp. vinsonii, and B. vinsonii subsp. berkhoffii. No evidence of a B. bacilliformis homolog of the 17-kDa antigen gene was obtained using multiple primer pairs. DNA sequencing revealed open reading frames capable of coding for proteins with sizes similar to that of the 17-kDa antigen of B. henselae in all of the amplicons; however, extensive sequence divergence across the genus was noted. Cloning of the amplified products into pUC19 resulted in recombinants that directed synthesis of homologs of the 17-kDa protein. Immunoblot analysis using human sera from CSD cases demonstrated very little cross-reactivity among different species for this protein. In contrast, immunoblots using rabbit serum raised to the recombinant B. henselae antigen showed extensive cross-reactivity with the proteins of other Bartonella species. The data suggest that the use of the 17-kDa antigen as a serologic reagent may allow the development of more specific diagnostic assays. Furthermore, the nucleotide sequences from the various versions of the 17-kDa antigen gene should be useful for rapid identification of Bartonella at the species level.