Introduction

Bartonella species are fastidious alpha-proteobacteria with worldwide distribution. To date, at least 15 species have been associated with human infections, with at least eight species also capable of infecting dogs and cats. Several blood-sucking arthropods have been suggested or confirmed as vectors for this genus, including sandflies, body lice, fleas, ticks, and keds [1]. In humans, select species of Bartonella were confirmed as etiologic agents of cat scratch disease (CSD), trench fever, bacillary angiomatosis and Oroya fever [2]. Moreover, Bartonella infections have also been documented by culture or molecular methods in human cases of endocarditis, myocarditis, polyarthritis and granulomatous inflammatory disease [1, 2]. In countries throughout the world, most diseases associated with Bartonella species infection are not reportable; therefore, incidence data is scarce. One study performed at the end of the 20^th century estimated that 22,000 new cases of cat scratch disease appear every year in the United States, and roughly 10% of these infections were thought to require hospitalization [3].

Bartonella species cause chronic and intermittent intra-erythrocytic bacteremia and infect endothelial cells of both incidental and natural reservoir hosts. The establishment of chronic, stealth infection is achieved by evasion of innate immune responses. These include resistance to complement activation, antigenic variation of surface proteins, and inhibition of host cell apoptosis [4]. Consequently, subclinical bloodstream infection in humans has been reported [5], supporting the fact that these bacteria will not follow Koch’s postulates for disease causation [6].

Asymptomatic Bartonella species infection poses a hazard for blood recipients because fast, sensitive and specific diagnostic tests are not currently available for donor screening. To the authors’ knowledge, no prophylactic measures are currently in place to prevent collection and transfusion of blood and blood products contaminated with species of Bartonella worldwide. Recently, using a combination of culture methods and PCR assays, we documented Bartonella henselae and Bartonella clarridgeiae infection of blood samples from 16 asymptomatic blood donors at a large blood bank center in Southeast Brazil [5]. Risk factors for Bartonella species exposure in blood donors were initially evaluated in one study from Sweden using serology methods [7]. However, antibody detection has limited predictive value in confirming or excluding Bartonella species bacteremia in humans and animals [6, 8–10]. Furthermore, higher vector activity is expected in tropical and sub-tropical regions of the world. Therefore, this study objective was to determine which risk factors are associated with Bartonella species infection in asymptomatic blood donors from a population in Campinas, Sao Paulo State, Brazil.

Methods

Results

Bartonella species bloodstream infection was detected in 16/500 blood donors (3.2%). DNA amplification and sequencing identified B. henselae in 15 blood donors (3%) and B. clarridgeiae in one donor (0.2%), which was previously reported [5, 13]. B. henselae bacteremia was also confirmed in six donors by bacterial isolation. The univariate and multivariate analyses of risk factors between blood donors infected with Bartonella species and uninfected subjects are provided in Tables 1 and 2. With univariate analysis, a professional with animal exposure was seven times more likely to be infected with Bartonella species than blood donors working in all other professions. When significant variables were entered into the multivariate logistic regression model, collinearity between animal-related professions and cat contact was documented; therefore, profession was not maintained in the final model. Adjusted odds ratio indicated that subjects with cat contact, or past history of tick bite, were approximately 3 to 4 times more likely to have a Bartonella species infection than donors without cat contact or lack of history of tick bite (Table 2).

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Table 1. Univariate analysis of risk factors between blood donors with Bartonella infection detected by enrichment PCR, compared with uninfected subjects^a.

https://doi.org/10.1371/journal.pntd.0004509.t001

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Table 2. Factors associated with occurrence of Bartonella infection in blood donors in Campinas, Brazil.

https://doi.org/10.1371/journal.pntd.0004509.t002

Discussion

This study identified two risk factors associated with subclinical Bartonella species bloodstream infections in blood donors. Bartonella species infection was three times more likely to be diagnosed in blood donors who had contact with cats compared to blood donors with no contact. Similarly, blood donors working in animal-related professions were seven times more likely to be infected with these pathogenic bacteria, although this variable was not included in the multivariate model due to collinearity. In Swedish blood donors, contact with cats was also a risk factor for B. elizabethae seropositivity [7]. Our findings indicate that cat contact also increases the risk of subclinical bloodstream infections with B. henselae or B. clarridgeiae. Previously, 24% of 192 non-immunocompromised Americans with frequent exposure to cats, cat scratches or fleas had detectable Bartonella species DNA in blood specimens using the same diagnostic approach as used in our study [9]. Cats are natural reservoir hosts for B. henselae, B. clarridgeiae, and Bartonella koehlerae, all of which are important zoonotic species [2]. After flea-transmitted infection with most Bartonella species strains, cats rarely develop clinical manifestations but remain persistently infected with high levels of intravascular bacteria, which facilitates pathogen acquisition by blood-sucking vectors [10]. Recently, artificial feeding of Ctenocephalides felis with B. henselae- or B. clarridgeiae- infected blood demonstrated that these pathogens can persist in C. felis and be excreted in flea feces [14]. Cat nails can be contaminated with infected flea feces, where B. henselae can survive for several days [15]. Needle stick transmission of Bartonella species has also been reported [16]. Bacterial transmission is less likely to occur by cat bite, since shedding of Bartonella species in cat saliva has not been clearly documented [17]. Interestingly, a history of cat bite, cat scratches, or flea bite was not significantly associated with Bartonella species infection in our study. Possible explanations include donors avoiding cat bites and scratches during “rough play”, lack of identifying fleas as the source of an insect bite, cat confinement to the house, and routine use of parasiticides.

In Southeast Brazil, the combined Bartonella prevalence reported in domestic and stray cats by five reports was 38.5% (112/291), but ranged from 4.3% to 97% among feline populations tested [18, 19, 20, 21, 22]. Such wide variation may be associated with level of flea infestation or analytical sensitivity of PCR assays used. B. henselae and B. clarridgeiae were the only two species detected by molecular methods from cats in these studies. Furthermore, we have tested 112 cats from the same county of the blood donors (Campinas, Brazil) for Bartonella bacteremia, using the same diagnostic approach used in the present report (BAPGM culture and PCR), where we detected bacteremia in 90% (101/112) of cats and B. henselae was confirmed by DNA sequencing and BLAST analysis from three culture isolates [23]. Therefore, similar to reports from other countries, cats may be the main reservoirs of B. henselae and B. clarridgeiae in Southeast Brazil, and pose as a risk factor for subclinical infection in humans in this region.

A previous history of tick bite was also determined to be a risk factor for Bartonella species infection in this human population. While cat fleas are a well-established vector, the capability of ticks to transmit these organisms has been the subject of substantial debate. It has been demonstrated that seven Bartonella species are capable of growing in an Amblyomma americanum tick cell line [24]. Recently, the vector competency of Ixodes ricinus for transmission of Bartonella birtlesii was confirmed using a mouse model [25]. Although. Ixodes ricinus and Amblyomma americanum are not present in Brazil, other ticks such as Ixodes loricatus, Ixodes didelphidis, Amblyomma cajennense, Amblyomma aureolatum, Amblyomma ovale, and Rhipicephalus sanguineus have been isolated from marsupials and rodents living in a locality where other tick-borne organisms had been previously identified [26]. Therefore, while the mode of transmission of any Bartonella species to humans is still unclear, the potential role of tick transmission should not be ignored.

Cat-scratch disease, bacillary angiomatosis and endocarditis are the most frequently reported manifestation of Bartonella infection in both immunocompetent or immunocompromised Brazilian populations [27–29], although ocular, neurologic and dermatologic abnormalities were also reported [30]. In addition, Correa et al. reported that the risk of Bartonella DNA bloodstream infection was 45 times higher in arrhythmic patients from Brazil or Argentina when compared to controls [31]. Our results expand the current understanding about Bartonella species infection in humans in Brazil by reinforcing the ecological role of cats and ectoparasites in the transmission of Bartonella. Risk of human infection can be minimized by implementing year-round ectoparasite control in domestic animals, by avoiding cat bites and scratches, and by keeping cats indoors to minimize exposure to vectors [32]. The medical impact of Bartonella species occurrence in the blood supply is still unknown and should be carefully investigated.

Supporting Information

Acknowledgments

We thank the study blood donors, Dr. Steven Henriksen and Dr. Dominique Griffon for providing research infrastructure and support at Western University of Health Sciences, and Dr. Hannah Mirrashed, John Greenwood, and Marizete Salvadego for technical assistance.