Bartonella species cause serious human infections globally, including bacillary angiomatosis, Oroya fever, trench fever, and endocarditis. We describe a patient who had fever and splenomegaly after traveling to Peru and also had bacteremia from an organism that resembled Bartonella bacilliformis, the causative agent of Oroya fever, which is endemic to Peru. However, genetic analyses revealed that this fastidious bacterium represented a previously uncultured and unnamed bartonella species, closely related to B. clarridgeiae and more distantly related to B. bacilliformis. We characterized this isolate, including its ability to cause fever and sustained bacteremia in a rhesus macaque. The route of infection and burden of human disease associated with this newly described pathogen are currently unknown.
Case Report
A 43-year-old American woman had a fever after traveling in Peru for 3 weeks. She visited Lima for several days, and then traveled to Nazca, where she resided in a lodge in a desert area at sea level. She then traveled to the Sacred Valley of Urubamba, followed by Cuzco and Machu Picchu, where she hiked and spent one night. Her trip concluded in the Amazon Basin near Iquitos. She received numerous insect bites, predominantly on the legs and feet.
Sixteen days after returning to the United States, the patient had fever, insomnia, myalgia, nausea, headache, and mild cough. During the first 4 days of fever, her temperature was as high as 38.9°C; it decreased during the next 3 days, but the day before presentation she had a recurrent fever, with a maximum temperature of 38.9°C. Six days before presentation she had a diffuse macular rash. She came to the clinic 8 days after the onset of fever. Physical examination revealed a temperature of 37.3°C, an enlarged spleen palpated 4 to 5 cm below the left costal margin, and healing insect bites on the legs and feet. Microscopical examination of a peripheral-blood specimen did not show bacteria or malaria parasites within erythrocytes. Laboratory values revealed a mild anemia that resolved 6 weeks after treatment (Table 1). A blood specimen from the patient was cultured, and she was given oral levofloxacin for 5 days as empirical treatment for enteric fever. One week later, she was asymptomatic and afebrile, and the spleen was not palpable. The patient did not own pets and had not been exposed to cats during or after her travel to Peru. There was no other recent travel, and her traveling companion remained well.
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A blood specimen from the patient was cultured in a 40-ml culture bottle (BACTEC Standard/10 Aerobic/F, Becton Dickinson) at the time of presentation. On day 15 of incubation, the automated culture system signaled a positive culture. Samples were submitted to one laboratory for Gram's staining, acridine orange staining, and subculturing and to another for subculturing. A 5-ml aliquot of blood was centrifuged at 5000xg for 45 minutes. The supernatant and pellet were plated onto heart infusion agar containing 5% fresh defibrinated rabbit blood2 and chocolate agar,7 and the plates were incubated at 28°C or 35°C in a candle-extinction jar. Fluorescence microscopy of the positive blood-culture broth stained with acridine orange was performed with the use of an epifluorescence microscope (excitation wavelength, 450 to 490 nm; mean emission wavelength, >515 nm). Subsequently, the morphologic characteristics of the isolate were examined by means of transmission electron microscopy after negative staining.8
On the basis of the presumptive identification of the isolate (designated BMGH) as a bartonella species, serum samples collected at presentation and follow-up were also assayed, with the use of class-specific conjugates labeled with fluorescein isothiocyanate, to determine reactivity against the isolate from the patient. In the IgM assays, samples were first depleted of IgG with the use of a protein-G removal device (Mini Rapi-Sep-M, PanBio). A reciprocal titer of 64 or more represents a positive result for B. henselae or B. quintana.9,10
Amplification by Polymerase Chain Reaction and Sequence Analysis
DNA from the BMGH isolate was extracted, and sequencing was performed as previously described.11,12,13,14,15 Oligonucleotide primers (from the Centers for Disease Control and Prevention Core Facility), gene targets, and GenBank accession numbers for nucleotide sequences generated in this study are listed in Table 1 of the Supplementary Appendix (available with the full text of this article at www.nejm.org). We aligned the sequences using ClustalW software and drew the phylogenetic tree using Molecular Evolutionary Genetics Analysis software, version 3.0.16 The distance matrix was calculated with the use of Kimura-2 parameters, and the tree was obtained with the use of the unweighted pair group method with arithmetic mean, based on a comparison of 1328 sites.
One-Dimensional and Two-Dimensional Gel Electrophoresis
For one-dimensional sodium dodecyl sulfate–polyacrylamide-gel electrophoresis (SDS-PAGE), bacterial proteins were electrophoresed on an 8 to 16% gradient gel and stained with Coomassie blue. For two-dimensional SDS-PAGE, subcellular fractions were prepared17,18 and a protease-inhibitor cocktail was added. Cytosolic proteins were precipitated and dialyzed overnight with the use of phosphate-buffered saline and then concentrated with a centrifuge filter (Amicon Ultra-4; Millipore). Two-dimensional gel electrophoresis was then performed19 (by Kendrick Laboratories): isoelectric focusing was carried out in glass tubes with the use of 2% pH 4–8 ampholines (BDH) for 9600 volt-hours. The tube gels were separated on 10% acrylamide slab gels and stained with silver nitrate.20
Inoculation of a Rhesus Macaque with the BMGH Isolate
To evaluate the ability of the isolate to cause disease, we next inoculated a rhesus macaque previously infected with the simian immunodeficiency virus. BMGH (culture passage 2) was grown for 6 days to confluency on 5% fresh defibrinated rabbit blood agar. The plates were scraped into M199 medium with Earle's salts and mixed, and 100 to 150 µl of the mixture (1.89x108 colony-forming units per milliliter) was inoculated intradermally in each of eight separate sites. In addition, 0.8 ml of inoculum was further diluted to a final volume of 4 ml with M199 medium with Earle's salts and was inoculated intravenously. Blood specimens from the inoculated macaque were cultured in a 2-ml EDTA tube (Becton Dickinson) twice a week and incubated at 35°C as previously described.3 All protocols and procedures were reviewed and approved by the institutional animal care and use committee.
Results
A bartonella-like bacterium was isolated from a blood specimen from the patient. Fifteen days after inoculation, a positive signal was detected in the BACTEC bottle inoculated with the blood specimen. No organisms were detected on Gram's staining, but clusters of organisms were found on acridine-orange staining (Figure 1A). Agar plates inoculated with broth from the blood culture with a positive signal grew confluent colonies after 10 days of incubation. Small, gram-negative bacilli with multiple, unipolar flagella were visualized on transmission electron microscopy (Figure 1B). The appearance of the BMGH isolate was indistinguishable from that of B. bacilliformis. Repeat blood cultures were performed 6 weeks after the first set, and the patient was treated with a second course of antibiotics, oral clarithromycin for 10 days. The follow-up cultures were negative.
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Comparison of the gene sequences of the 16S ribosomal DNA (rDNA), the gltA and rpoB fragments, and the 16S–23S intergenic spacer region showed that the BMGH isolate is a new bartonella species, most closely related phylogenetically to B. clarridgeiae. The sequence of an 1171-bp fragment of the BMGH 16S rDNA had 99.2 to 99.7% similarity to that of B. clarridgeiae, with nine variable nucleotide positions (four that were consistently different from those found in the five B. clarridgeiae isolates tested) (Table 2 of the Supplementary Appendix).
Among the bartonella species, sequences of BMGH gltA and rpoB fragments were again most similar to B. clarridgeiae (95.9% and 91.7% similarity, respectively) (Table 3 of the Supplementary Appendix), a finding that is consistent with the proposed criteria for a new bartonella species.21 Searching with the use of the Basic Local Alignment Search Tool revealed that the 1439-bp fragment of the 16S–23S intergenic spacer region was most similar to that of the uncultured bartonella F17688 [GenBank] (GenBank accession no. AF415211 [GenBank] ), amplified from a flea (pulex species) removed from a human in Cuzco, Peru.22 The 970-bp sequence available for bartonella F17688 [GenBank] had 99.8% similarity with the corresponding sequence of the 16S–23S intergenic spacer region of the BMGH isolate (with only two differences detected). Phylogenetic analysis of this fragment resulted in the placement of the BMGH isolate and the uncultured bartonella F17688 [GenBank] in the same clade as B. clarridgeiae (Figure 2), but the eight B. clarridgeiae isolates from different geographic regions formed a phylogenetic lineage that differed from that of the BMGH and F17688 [GenBank] isolates (supported by 100% of the bootstrap replications). These genetic data indicate that BMGH is a new bartonella species, which we have named B. rochalimae, that is distinct from B. clarridgeiae.
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After experimental inoculation of the macaque with BMGH, bacteremia was detected first on day 14 and also on days 17, 22, and 24 (0.75, 1.50, 12.75, and 11.25 colony-forming units per milliliter, respectively). Isolates were confirmed to be BMGH by means of amplification and sequencing of the 16S rDNA. The animal had an increase in temperature (from 37.6°C to 38.7°C on day 3) and a decrease in hematocrit (from 40.1% to 33.9% on day 7, with a gradual increase to 38.3% by day 70).
Discussion
The bartonella genus consisted of a single species in 1992, but there are now 19 officially recognized and extant species and subspecies. At present, humans are the sole reservoir host for only two species of bartonella: B. quintana and B. bacilliformis. One additional species, B. henselae, has been isolated occasionally from immunocompromised humans, but the domestic cat is the primary mammalian reservoir for this species. Distinct epidemiologic risk factors are significantly associated with the acquisition of these three bartonella species that infect humans: homelessness and exposure to human body lice for B. quintana,4 scratches from kittens infested with fleas for B. henselae,23 and visits to or residence in a geographically restricted region of the Andes where the sand-fly vector is found for B. bacilliformis.24
Our patient presented with fever, splenomegaly, anemia, and a history of insect bites after a trip to Peru that included travel to a region in which B. bacilliformis is endemic. On the basis of clinical, epidemiologic, and microbiologic evidence, we initially assumed that the small, motile, fastidious bacterium isolated from her blood was B. bacilliformis. However, molecular characterization of the organism revealed a new species of bartonella, closely related to, but distinct from, B. clarridgeiae. The mammalian reservoir for B. clarridgeiae is the domestic cat, and the prevalence of B. clarridgeiae bacteremia in cats varies worldwide, from less than 10% in the United States to 36% in Europe.25,26 Despite the substantial prevalence of B. clarridgeiae in cats, it has never been isolated in a human or amplified from human tissue specimens, including those from patients with cat scratch disease or bacillary angiomatosis.4,27
B. bacilliformis has caused multiple outbreaks of Oroya fever in persons living in certain regions of Peru, Ecuador, and Colombia. Many of these persons received a diagnosis of infection with B. bacilliformis on the basis of clinical findings and examination of blood smears or blood cultures. Although we did not observe intraerythrocytic organisms on the blood smear from our patient, the fever, anemia, splenomegaly, and isolation of a bartonella species raise the possibility that some cases of Oroya fever could represent infection with this new bartonella species. It will be of interest to determine the prevalence of this new species in humans residing in the Andes and to identify whether it also can cause a form of infection similar to verruga peruana, the chronic form of infection with B. bacilliformis.
B. bacilliformis and B. quintana are transmitted to humans by arthropods. Our patient noted insect bites on her legs and feet during her trip to Peru, but the source insect could not be identified. Using a polymerase-chain-reaction assay, Parola et al.22 identified a sequence for the 16S–23S intergenic spacer region in a pulex flea found on a person in Cuzco, Peru, that was nearly identical to the BMGH sequence — suggesting a possible vector.
In conclusion, we have identified a new bartonella species that caused an illness with features resembling Oroya fever in a patient who had recently traveled to Peru. Whether a zoonotic reservoir exists and the mechanism by which human infection occurs are currently unknown. This case illustrates the importance of culturing specimens from patients to test for bartonella species, performing careful molecular characterization of bartonella isolates, and remaining vigilant for new bartonella species that are pathogenic in humans.
To fulfill the rules of the International Code of Nomenclature of Bacteria [Lapage SP, Sneath PHA, Lessel EF, Skerman VBD, Seeliger HPR, Clark WA. International code of nomenclature of bacteria (1990 revision). Washington, DC: American Society for Microbiology, 1992.], we provide the following description of the novel species identified in this report. Description of Bartonella rochalimae sp. nov. Bartonella rochalimae (ro•cha•li•ma'e. N.L. gen. masc. n. rochalimae, of Rochalima, named in honor of Henrique da Rocha-Lima, an early Brazilian investigator of the etiology of rickettsial diseases). Small, motile, fastidious gram-negative rod with multiple, unipolar flagella that grows on fresh defibrinated rabbit-blood agar at 35°C in a candle extinction jar. Bartonella rochalimae is differentiated genetically from other Bartonella species on the basis of unique sequences of 16S rDNA, gltA and rpoB genes, and 16S–23S intergenic spacer region. The bacterium infects and is pathogenic for humans and macaques. The type strain is BMGH (available as American Type Culture Collection no. BAA-1498), isolated from a 43-year-old woman with splenomegaly, fever, anemia, and recent travel to Peru.
Supported by grants from the National Institutes of Health (R01 AI43703 and R01 AI52813), a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research (to Dr. Koehler), and core funding to the Rickettsial Zoonoses Branch of the Centers for Disease Control and Prevention.
No potential conflict of interest relevant to this article was reported.
We thank Nafisa Ghori for her assistance with the electron microscopy and Wilhelm von Morgenland, Linda Hirst, and the Research Services Staff for their technical assistance with the primate inoculation at the California National Primate Research Center, University of California at Davis.
Source Information
From the Centers for Disease Control and Prevention, Atlanta (M.E.E., S.L.L., W.L.N., G.A.D.); University of California at San Francisco, San Francisco (H.L.G., J.S.G., S.S.M., J.E.K.); and Massachusetts General Hospital and Harvard Medical School — both in Boston (E.T.R., M.J.F., J.M.H.).
Address reprint requests to Dr. Koehler at the Division of Infectious Diseases, Box 0654, University of California at San Francisco, San Francisco, CA 94143-0654.
References
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