Case Report
Bartonella spp. bacteremia in high-risk immunocompetent patients,☆☆,

https://doi.org/10.1016/j.diagmicrobio.2011.09.001Get rights and content

Abstract

Serum and blood samples from 192 patients, who reported animal exposure (100.0%) and recent animal bites or scratches (88.0%), were screened for antibodies by indirect immunofluorescence assays and for bacteremia using the BAPGM (Bartonella alpha Proteobacteria growth medium) platform. Predominant symptoms included fatigue (79.2%), sleeplessness (64.1%), joint pain (64.1%), and muscle pain (63.0%). Bartonella spp. seroreactivity or bacteremia was documented in 49.5% (n = 95) and 23.9% (n = 46) of the patients, respectively; however, indirect immunofluorescence antibodies were not detected in 30.4% (n = 14) of bacteremic patients. Regarding components of the BAPGM platform, Bartonella DNA was amplified from 7.5% of blood (n = 21), 8.7% of serum (n = 25), and 10.3% of enrichment culture samples (n = 29). Polymerase chain reaction (PCR) on only extracted blood would not have detected Bartonella infection in 34.7% (16/46) of bacteremic patients. Serology, in conjunction with blood, serum, and BAPGM enrichment culture PCR, facilitates the diagnosis of Bartonella spp. bacteremia in immunocompetent patients.

Introduction

Bacteria of the genus Bartonella (alpha subdivision of the class Proteobacteria) are fastidious, Gram-negative, aerobic bacilli with more than 26 described species or subspecies (Boulouis et al., 2005, Boulouis and Chomel, 2004, Breitschwerdt et al., 2007a, Breitschwerdt et al., 2007b, Breitschwerdt et al., 2010b, Chomel et al., 2004, Chomel et al., 2006, Houpikian and Raoult, 2005, Joblet et al., 1995, Maurin et al., 1997). Seventeen Bartonella spp. have been associated with an expanding spectrum of human diseases, ranging from acute fever, to more severe disease manifestations including encephalopathy, endocarditis, sensory and motor neuropathies, pleural and pericardial effusion, pneumonia, and hemolytic anemia. Bartonella are highly adapted to various distinct mammalian reservoir hosts, including domestic animals, human beings, marine mammals, rodents, and ungulates, wherein these bacteria cause long-lasting intra-erythrocytic and intravascular infection, which can be associated with a relapsing pattern of bacteremia, as demonstrated in cats and rodents (Chomel et al., 2003, Dehio, 2001, Houpikian and Raoult, 2005, Joblet et al., 1995, Jones et al., 2008, Kordick and Breitschwerdt, 1995, Kordick and Breitschwerdt, 1998, Maggi et al., 2009). Conventional microbiological approaches for documentation of Bartonella infections, including ELISA, immunofluorescence antibody assays, and, because of the fastidious nature of these bacteria, isolation on agar plates, all remain relatively insensitive and of limited diagnostic utility, particularly when only 1 test is used. When infection cannot be confirmed by polymerase chain reaction (PCR), diagnostic confirmation has often relied on a combination of clinical (fever), epidemiological (history of cat scratch), and serologic criteria (Breitschwerdt et al., 2009, Maggi et al., 2006). In conjunction with efforts to improve the diagnostic documentation of Bartonella bacteremia, our laboratory described a novel, chemically modified, insect cell culture–based liquid growth medium, Bartonella alpha Proteobacteria growth medium (BAPGM), that experimentally facilitated growth of at least 7 Bartonella species (Maggi et al., 2006). Subsequently, we used BAPGM enrichment culture to increase the levels of bacteria in patient samples, so as to enhance detection by PCR amplification or by subculture isolation of a Bartonella sp. onto blood agar plates (Breitschwerdt et al., 2007a, Breitschwerdt et al., 2007b, Breitschwerdt et al., 2008, Maggi and Breitschwerdt, 2005, Maggi et al., 2005). BAPGM pre-enrichment medium is formulated using the basal biochemical composition of the insect growth media IPL-41 (Sigma-Aldrich, St. Louis, MO, USA), which is supplemented with several amino acids, proteins, precursors of biosynthesis (vitamins), and other nutrients. Experimentally, BAPGM also supported the growth of co-cultures composed of 2 Bartonella sp., which subsequently lead to the microbiological documentation of co-infections in the blood of dogs and human patients (Breitschwerdt et al., 2007a, Breitschwerdt et al., 2007b, Breitschwerdt et al., 2008, Maggi and Breitschwerdt, 2005, Maggi et al., 2005). More recently and for the first time, investigators at the Centers for Disease Control and prevention (CDC) used BAPGM to facilitate the isolation of a new Bartonella sp. (Candidatus Bartonella tamiae) from febrile patients in Thailand (Kosoy et al., 2010). The same investigators subsequently used either BAPGM or cell culture enrichment to increase the quantity of Bartonella organisms prior to PCR amplification of Bartonella spp. DNA from the blood of dogs or febrile human patients in Thailand (Bai et al., 2010, Kosoy et al., 2008, Kosoy et al., 2010). Despite the ability of BAPGM to support the growth of Bartonella spp. (Maggi et al., 2005), cumulative microbiological results using this liquid enrichment blood culture approach in a large at-risk human population has not been previously reported.

Previous reports have estimated the Bartonella seroprevalence in healthy veterinarians at 7.1% (Noah et al., 1997). However, the prevalence of Bartonella spp. bacteremia in healthy or sick veterinary professionals who experience frequent animal bites and scratches or extensive arthropod exposure is currently unclear. In this study, we describe serologic and microbiological results using the BAPGM platform from a low-risk control group and from 192 patients who had experienced frequent animal contact and arthropod exposures, of whom 46 were bacteremic with 1 or more Bartonella sp. Indirect immunofluorescence antibody (IFA) assays targeting B. henselae, B. koehlerae, and B. vinsonii subsp. berkhoffii antigens were descriptively compared to PCR and DNA sequencing results obtained from whole blood, serum, BAPGM enrichment whole blood culture, and subculture isolates. In addition, we describe self-reported symptoms, historical exposures to animals and vectors, and previous clinical diagnoses.

Section snippets

Study population

Between September 2007 and June 2010, 192 patients with extensive arthropod exposure and/or frequent animal contact voluntarily entered into the study. Participants became aware of the study through lay publications and by attending lectures on canine and feline bartonellosis at regional and national veterinary conferences. In most instances, patients requested testing because of a history of chronic poorly defined illness, fatigue, joint pain, arthritis, and neurologic or neurocognitive

Serology

IFA serology results for B. vinsonii subsp. berkhoffii (genotypes I and III) and B. koehlerae were negative in all 32 control samples. One control was B. henselae seroreactive at a reciprocal titer of 64. Unexpectedly, 16 controls were B. vinsonii subsp. berkhoffii genotype II seroreactive at reciprocal titers of 64 to 512. Seroreactivity to genotype II antigens in the patient population was 28.1% (54/192).

BAPGM platform

All components (blood, serum, 7- and 14-day enrichment cultures, 4 PCRs per platform) of

Discussion

It is well recognized that Bartonella spp. are transmitted by various arthropod vectors and by animal bites and scratches (Boulouis et al., 2005, Boulouis and Chomel, 2004, Breitschwerdt et al., 2007a, Breitschwerdt et al., 2009, Chomel et al., 2004, Chomel et al., 2006). Based upon serologic testing using a panel of 5 antigens, exposure to Bartonella species was common (49.5%) among the study population and nearly a fourth of the patients were bacteremic, a subset of whom (30.4%) did not have

Acknowledgments

The authors would like to thank the physicians and patients who facilitated sample collection and submission to our laboratory for testing purposes and Tonya Lee for editorial assistance.

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    Clinical relevance: This manuscript describes clinical, serologic, and molecular findings in immunocompetent patients with extensive animal contact and arthropod exposure.

    ☆☆

    Supported in part by the state of North Carolina, a grant from the American College of Veterinary Internal Medicine Foundation, and a donation from Bayer Animal Health.

    Disclosure: In conjunction with Dr. Sushama Sontakke and North Carolina State University, Dr. Breitschwerdt holds U.S. patent no. 7,115,385 (Media and Methods for Cultivation of Microorganisms), which was issued October 3, 2006. He is the chief scientific officer for Galaxy Diagnostics, a newly formed company that provides diagnostic testing for the detection of Bartonella species infection in animals and in human patient samples. Dr. Ricardo Maggi has led efforts to optimize the BAPGM platform and is the scientific technical advisor and laboratory director for Galaxy Diagnostics.

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