Russell L. Regnery

Syndrome of Rochalimaea henselae adenitis suggesting cat scratch disease

Rochalimaea henselae is a causative agent of bacillary angiomatosis [1, 2] and peliosis hepatis [3, 4] in patients infected with human immunodeficiency virus (HIV). The organism has been isolated in culture from both immunocompromised and immunocompetent patients with fever and bacteremia [5, 6]. It has been associated with aseptic meningitis and, like R. quintana and Bartonella spp., can be associated with relapsing disease and with persistent bacteremia in the absence of symptoms [7-12]. Cat scratch disease has been provisionally associated with the fastidious gram-negative rod Afipia felis [13]; however, A. felis has only rarely been isolated from patients with cat scratch disease, and evidence of A. felis-specific antibodies is lacking in most persons with suspected cat scratch disease [14]. In contrast, nearly 90% of persons with suspected cat scratch disease have serologic evidence of Rochalimaea infection [14]. This disease typically presents as adenitis with an evident papular lesion at the inoculation site [15, 16]. Other clinical presentations of cat scratch disease include neurologic syndromes [17], liver disease [18], angiomatous skin lesions [19], and prolonged, recurrent infection [20]. Although several similarities exist between R. henselae infection and cat scratch disease in clinical presentation, the typical cat scratch disease syndrome of adenitis caused by R. henselae has not been described. We report two cases of otherwise healthy patients with upper-extremity adenitis in which R. henselae was isolated from the infected lymph nodes, drawing a parallel to the most common presentation of cat scratch disease and adding to the spectrum of disease caused by R. henselae. Case Reports Patient 1 A 68-year-old white man with a history of mild hypertension was seen in the emergency department; he complained of 72 hours of fever to 39.2 C and an enlarging mass at his left elbow. In the emergency department his temperature was 39.4 C and he had a small eschar in the interphalangeal space of his left hand between the third and fourth digits that he reported had been present for 2 months. His environmental exposures included small lacerations from rose bushes he tended and contact with a pet cat. His leukocyte count was 17.3 109/L with a predominance of neutrophils and band forms. The patient was given a 10-day course of cefadroxil. He became afebrile during therapy but because the left epitrochlear mass was enlarging and becoming painful, he again sought medical attention. At this time he had a small 1 0.5-cm nonhealing ulcer without eschar on the dorsum of the 3 to 4 interphalangeal space of the left hand and a 4 4-cm, tender, nonfluctuant left epitrochlear lymph node. No other adenopathy was present, and the rest of the examination was unremarkable. Laboratory test results, including complete blood count, chemistry profile with liver functions studies, urinalysis, and erythrocyte sedimentation rate, were normal. Result of a test for HIV-1 was negative. A chest roentgenogram was normal. Serologic test results for tularemia, brucellosis, and syphilis were negative. A purified protein-derivative skin test result was negative with positive controls. An excisional biopsy of both the interdigital lesion and the epitrochlear node was done. Histopathologic examination revealed similar processes in both sites characterized by necrotizing granulomas in a background of chronic inflammation, with multinucleated giant cells and a mixed perivascular infiltrate (Figure 1). No organisms were seen with tissue acid fast or Gram stain, Warthin-Starry stain, or Gomori-methenamine-silver stain. Figure 1. Photomicrographs of the papular hand lesion and lymph node of Patient 1. Top left. Top right. Bottom. A sample of the lymph node was cultured, and an organism was isolated that was identified as R. henselae. The patient was treated with a 28-day course of doxycycline and ciprofloxacin. The lesion healed after 10 days, and he has remained symptom free for 9 months. Patient 2 A previously healthy Hispanic 27-year-old man developed a tender left axillary lymph node while traveling through Hawaii, Thailand, Guam, and Bangladesh as a flight crew member. Three days later, he developed fevers to 38.9 C, light-headedness, fatigue, and nausea with one episode of vomiting, chills, diaphoresis, and headache. The systemic symptoms resolved after 3 days, but the node continued to enlarge and became more painful. When we examined him, he was afebrile and complained only of the painful node that limited use of his left arm. The patient owned and handled cats, dogs, and goats and had received tick bites, although not on the affected limb. He had a 4 5-cm nonfluctuant, tender left axillary lymph node with minimal overlying erythema, a 1 1-cm left epitrochlear lymph node that was nontender, and a 1 1-cm eschar on the palmar surface of his left hand. The examination was otherwise unremarkable. Values were normal for a complete blood count, serum chemistry tests, urinalysis, and erythrocyte sedimentation rate. Serologic test results for tularemia, brucellosis, plague, syphilis, and scrub typhus were negative, as was a test for HIV-1. The purified protein derivative skin test was negative with positive controls. An excisional biopsy of the axillary lymph node was done, revealing hard, inflamed, adherent, matted nodes at surgery. The patient was given a 14-day course of cephradine followed by a 28-day course of doxycycline after surgery. Histopathologic examination of the lymph node sample showed findings similar to those of the skin and lymph node biopsies of Patient 1. Cultures of the biopsy material grew rare, small colonies of R. henselae after a prolonged incubation. Concurrent cultures of the blood remained negative after 30 days. The patient recovered and has remained symptom free for 12 months. Methods Microbial Isolation Biopsy material was placed on saline-soaked gauze and carried directly to the microbiology laboratory, where it was ground in a tissue grinder and plated onto chocolate and CDC anaerobic blood agar (sheep blood with added hemin and L-cysteine; BDMS, Cockeysville, Maryland), Jem Bec plates (BDMS) and placed into liquid phase media, including cooked meat broth (BDMS) and eugonic broth (modified, Remel, Lexena, Kansas). Fungal and mycobacterial cultures were also done. These materials were then incubated at 35 C in 5% CO2, with plates kept upright for 24 hours and then inverted. Plates were incubated for up to 6 weeks under these conditions to optimize the isolation of fastidious organisms. For determination of X and V factor dependence, cultures were plated onto brain-heart infusion agar with X, V, and XV strips (BDMS). Broth Microdilution Antimicrobial Susceptibility Tests The two R. henselae isolates described above and a previously described blood isolate [7] were tested for susceptibility to a wide array of antimicrobial agents by a broth microdilution susceptibility method analogous to that used to test Haemophilus influenzae. Specifically, microdilution trays were prepared with twofold concentration increments of the various antimicrobial agents incorporated into Haemophilus test medium [21, 22] dispensed into 100-L/well aliquots. The bacterial inoculum was prepared from growth of the test strains that had been isolated from a single colony and subcultured on enriched chocolate agar and incubated for 5 to 7 days at 35 C in 5% CO2. The final inoculum density used for all susceptibility tests was approximately 5 105 colony-forming units per mL derived by suspending growth in 0.9% NaCl to a turbidity of 0.5 McFarland standard. Plate counts verified the appropriateness of the final inoculum. Microdilution trays were incubated for 7 days at 35 C in ambient air before minimum inhibitory concentrations were interpreted in the usual manner. Whole-Cell Fatty Acid Analysis Analysis of whole-cell fatty acids was done with a Hewlett Packard 5890 gas chromatography system and software (Microbial Identification System, Version 3.0, Microbial ID, Inc., Newark, Delaware). Cultures of R. henselae, R. quintana, and A. felis (strain B.V., Armed Forces Institute of Pathology, Washington, DC) were grown for 7 days on CDC blood agar at 35 C in increased CO2, and then colonies were harvested with a loop. The cellular material was then saponified, methylated, extracted, base-washed, and analyzed as recommended by the manufacturer. DNA Analysis The identification of the bacteria was confirmed by using a combination of the polymerase chain reaction (PCR) amplification of the citrate synthetase gene sequence and subsequent restriction fragment length polymorphism analysis of the resultant amplified DNA product [6, 23]. The PCR-amplified DNA was subjected to restriction-endonuclease digestion with various enzymes (HinfI, HhaI, MseI, and TaqI). The sizes of resulting DNA fragments were compared by polyacrylamide gel electrophoresis with DNA fragments prepared simultaneously from other Rochalimaea strains, including R. quintana (ATCC VR-358, isolate Fuller), R. vinsonii (ATCC VR-152, vole agent), and R. henselae, (Houston-1 prototype isolate). Results Colonies were noted in the area of application on the CDC anaerobic blood plates at day 33 and day 13 in the first and second cases, respectively. Growth was slower on chocolate agar and did not occur on the other media used. Afipia felis (strain B.V.) was subcultured onto blood agar under identical conditions to show that Afipia could be grown using the method described here to isolate R. henselae. Rochalimaea colonies were small, nonhemolytic, rough and dry, and yellow to grey in color. Gram stain revealed small, curved, pleomorphic gram-negative rods. The organism was oxidase and catalase negative and X-factor dependent. Growth occurred more quickly on subsequent subcultures. The most rapid growth was attained using human blood agar. Rochalimaea quintana has previously been shown to grow bet

Rats of the genus Rattus are reservoir hosts for pathogenic Bartonella species: an Old World origin for a New World disease?

Bartonella species were isolated from the blood of 63 of 325 Rattus norvegicus and 11 of 92 Rattus rattus from 13 sites in the United States and Portugal. Infection in both Rattus species ranged from 0% (e.g., 0/87) to approximately 60% (e.g., 35/62). A 337-bp fragment of the citrate synthase (gltA) gene amplified by polymerase chain reaction was sequenced from all 74 isolates. Isolates from R. norvegicus were most similar to Bartonella elizabethae, isolated previously from a patient with endocarditis (93%-100% sequence similarity), followed by Bartonella grahamii and other Bartonella species isolated from Old World rodents (Clethrionomys species, Mus musculus, and Rattus species). These data suggest that Rattus species are a reservoir host for pathogenic Bartonella species and are consistent with a hypothesized Old World origin for Bartonella species recovered from Rattus species introduced into the Americas.

Severe Eczema Vaccinatum in a Household Contact of a Smallpox Vaccinee

BACKGROUND We report the first confirmed case of eczema vaccinatum in the United States related to smallpox vaccination since routine vaccination was discontinued in 1972. A 28-month-old child with refractory atopic dermatitis developed eczema vaccinatum after exposure to his father, a member of the US military who had recently received smallpox vaccine. The father had a history of inactive eczema but reportedly reacted normally to the vaccine. The childs mother also developed contact vaccinia infection. METHODS Treatment of the child included vaccinia immune globulin administered intravenously, used for the first time in a pediatric patient; cidofovir, never previously used for human vaccinia infection; and ST-246, an investigational agent being studied for the treatment of orthopoxvirus infection. Serological response to vaccinia virus and viral DNA levels, correlated with clinical events, were utilized to monitor the course of disease and to guide therapy. Burn patient-type management was required, including skin grafts. RESULTS The child was discharged from the hospital after 48 days and has recovered with no apparent systemic sequelae or significant scarring. CONCLUSION This case illustrates the need for careful screening prior to administration of smallpox vaccine and awareness by clinicians of the ongoing vaccination program and the potential risk for severe adverse events related to vaccinia virus.

Monkeypox transmission and pathogenesis in prairie dogs.

During May and June 2003, the first cluster of human monkeypox cases in the United States was reported. Most patients with this febrile vesicular rash illness presumably acquired the infection from prairie dogs. Monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. Immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with less amounts in adjacent macrophages, fibroblasts, and connective tissues. Viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. Virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. These findings indicate that both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus between rodents and to humans. Prairie dogs offer insights into transmission, pathogenesis, and new vaccine and treatment trials because they are susceptible to severe monkeypox infection.

A prairie dog animal model of systemic orthopoxvirus disease using West African and Congo Basin strains of monkeypox virus.

Multiple monkeypox virus (MPXV) animal models have been discussed in previous studies, but no small animal models, nor most non-human primate models, demonstrated the protracted asymptomatic incubation phase seen in systemic human orthopoxvirus illness. Herein, we characterize a black-tailed prairie dog (PD) (Cynomys ludovicianus) model of infection, via intranasal and intradermal exposures, with the two MPXV clades. Daily observations of the animals were made (food consumption, general symptoms, disease presentation), while weights and virus evaluations (ocular, nasal, oropharyngeal, faeces, blood) were obtained/made every third day. Generalized rash became apparent 9-12 days post-infection for all animals. Individual animals demonstrated a range of symptoms consistent with human monkeypox disease. Measurable viraemias and excretas were similar for both clade-representative strains and persisted until at least day 21. Greater morbidity was observed in Congo Basin strain-challenged animals and mortality was observed only in the Congo Basin strain-challenged animals. The PD model is valuable for the study of strain-dependent differences in MPXV. Additionally, the model closely mimics human systemic orthopoxvirus disease and may serve as a valuable non-human surrogate for investigations of antivirals and next generation orthopoxvirus vaccines.

Evaluation of intraspecies genetic variation within the 60 kDa heat-shock protein gene (groEL) of Bartonella species.

A phylogenetic investigation was done on the members of the genus Bartonella, based on the DNA sequence analysis of the groEL gene, which encodes the 60 kDa heat-shock protein GroEL. Nucleotide sequence data were determined for a near full-length fragment (1368 bp) of the groEL gene of the established Bartonella species and used to infer intraspecies phylogenetic relationships. Phylogenetic trees were inferred from multiple sequence alignments by using both distance and parsimony methods, which demonstrated an architecture composed of six well-supported lineages. The results are consistent with relationships deduced from recent sequence analysis studies based upon citrate synthase (gItA) and previously observed genotypic and phenotypic characteristics; however, they showed greater statistical support at the intragenus level. This suggests that groEL may be a more robust tool for phylogenetic analysis of Bartonella lineages.

Cluster of five children with acute encephalopathy associated with cat-scratch disease in South Florida

Between August 12 and September 27, 1994, five children in South Florida were hospitalized at a single hospital because of encephalopathy, presenting as status epilepticus, associated with cat-scratch disease (CSD). Diagnoses were confirmed by using an indirect fluorescent antibody test to detect antibody to Bartonella henselae, the causative agent of CSD. These cases represent the first cluster of CSD encephalopathy cases to be recognized in the United States. The patients lived within 7 miles of each other and all reported contact with pet or stray cats before developing regional lymphadenopathy and encephalopathy. All recovered fully. A high proportion of 124 cats from the local area were seropositive (62%) or bacteremic (22%). This study suggests that B. henselae can be associated with geographically focal clusters of CSD encephalitis and should be considered in the evaluation of children with acute encephalopathy.

ISOLATION OF BARTONELLA SPP. FROM EMBRYOS AND NEONATES OF NATURALLY INFECTED RODENTS

Embryos and neonatal offspring of wild-captured cotton rats (Sigmodon hispidus) and white-footed mice (Peromyscus leucopus) were tested for the presence of Bartonella spp. Isolates of Bartonella spp. were obtained from 18 of 31 embryos and 7 of 19 neonates from bacteremic dams of the two species; no isolates were obtained from material from non-bacteremic dams. Sequence analysis demonstrated that the isolates from embryos and neonates matched the phylogenetic group of Bartonella spp. isolates obtained from the mother. No antibodies to homologous Bartonella spp. antigens were detected in maternal and neonatal blood or embryonic tissue. These findings suggest the possibility of vertical transmission of Bartonella spp. among natural rodent hosts.

A silent enzootic of an orthopoxvirus in Ghana, West Africa: evidence for multi-species involvement in the absence of widespread human disease.

Human monkeypox has never been reported in Ghana, but rodents captured in forested areas of southern Ghana were the source of the monkeypox virus introduced into the United States in 2003. Subsequent to the outbreak in the United States, 204 animals were collected from two commercial trapping sites in Ghana. Animal tissues were examined for the presence of orthopoxvirus (OPXV) DNA using a real-time polymerase chain reaction, and sera were assayed for antibodies against OPXV. Animals from five genera (Cricetomys, Graphiurus, Funiscirus, and Heliosciurus) had antibodies against OPXV, and three genera (Cricetomys, Graphiurus, and Xerus) had evidence of OPXV DNA in tissues. Additionally, 172 persons living near the trapping sites were interviewed regarding risk factors for OPXV exposure, and their sera were analyzed. Fifty-three percent had IgG against OPXV; none had IgM. Our findings suggest that several species of forest-dwelling rodents from Ghana are susceptible to naturally occurring OPXV infection, and that persons living near forests may have low-level or indirect exposure to OPXV-infected animals, possibly resulting in sub-clinical infections.

Ebola virus: identification of virion structural proteins.

Polyacrylamide gel electrophoresis of purified Ebola virus revealed the presence of four major virion structural proteins which we have designated VP1, VP2, VP3 and VP4. Vesicular stomatitis virus (VSV) proteins were used as mol. wt. markers, and the virion proteins were found to have mol. wt. of 125000 (VP1), 104000 (VP2), 40000 (VP3) and 26000 (VP4). VP1 was labelled with glucosamine and is probably a glycoprotein. The density of the Ebola virion was approx. 1.14g/ml in potassium tartrate. Virus nucleocapsids with a density of 1.32g/ml in caesium chloride were released when virions were treated with detergents. Proteins VP2 and VP3 were consistently associated with released nucleocapsids and are probably the major structural nucleocapsid proteins analogous to the N protein of VSV. Protein VP4 was reduced or absent in released nucleocapsids and is probably analogous to the membrane (M) protein of VSV and similar viruses. The glycoprotein (VP1) is larger than the glycoprotein of any known negative-strand RNA virus and is not labelled well with 35S-methionine. VP1 is solubilized by detergent treatment, suggesting that it is a component of the virion spikes and analogous to the G protein of VSV. Our results, in conjunction with analysis of Ebola virion RNA (Regnery et al. 1980), strongly suggest that the virus is a negative-strand RNA virus and, along with marburg virus, may constitute a new taxon within this group.