Bartonella species in Cambodia, Ghana, Laos, and Peru: results from vector and sero-surveys

Bartonella species are fastidious Gram negative vector-borne bacteria with a wide range of mammalian reservoirs. While it is understood that some species Bartonella are human pathogens, the extent of human exposure to Bartonella species (both pathogenic and non-pathogenic) has yet to be fully understood. To this end, residual sera from participants enrolled in undifferentiated fever studies in Cambodia, Ghana, Laos, and Peru were screened for the presence of IgG antibodies against B. quintana and B. henselae, using the FOCUS diagnostics Dual Spot- Bartonella IgG Immunofluorescence assay. Forty-eight patients with suspected or confirmed B. bacilliformis exposure or infection in Peru, were screened to assess cross-reactivity of the FOCUS assay for IgG against other Bartonella species. Ten of 13 patients with confirmed B. bacilliformis infection were Bartonella-specific IgG positive and overall, 36/48 of the samples were positive. Additionally, 79/206, 44/200, 101/180, and 57/100 of samples from Peru, Laos, Cambodia, and Ghana, respectively were Bartonella-specific IgG positive. Further, ectoparasites pools from Cambodia, Laos, and Peru were tested using quantitative real-time PCR (qPCR) for the presence of Bartonella DNA. Of the sand-fly pools collected in Peru, 0/196 were qPCR positive; 15/140 flea pools collected in Cambodia were qPCR positive; while 0/105 ticks, 0/22 fleas, and 0/3 louse pools collected in Laos tested positive for Bartonella DNA. Evidence of Bartonella in fleas from Cambodia supports the possibility that humans are exposed to Bartonella through this traditional vector. However, Bartonella species were not found in fleas, ticks, or lice from Laos, or sandflies from Peru. This could account for the lower positive serology among the population in Laos and the strictly localized nature of B. bacillformis infections in Peru. Human exposure to Bartonella species and Bartonella as a human pathogen warrants further investigation.


Introduction
Bartonella species are fastidious, Gram negative, vector-borne bacteria. Classically, three Bartonella species have been identified as causing human bartonellosis, B. bacilliformis, B. quintana, and B. henselae, however a growing number of Bartonella species are being implicated in human disease. The genus grew from just one member with the discovery of B. bacilliformis in 1909, to five members in 1993, when the Bartonella and Rochalimaea genera were united(R L Regnery, Anderson, et al. 1992;Noguchi and Battistini 1926;Brenner et al. 1993;Daly et al. 1993;Russell L Regnery, Olson, et al. 1992). Since then, the number of known Bartonella species has grown to more than fifty-five current and Candidatus members (Breitschwerdt 2017;K. E. Mullins et al. 2015;Mangombi et al. 2020;Gutiérrez et al. 2020;Medkour et al. 2019). Bartonella species have been isolated from a wide variety of invertebrates, including fleas, ticks, body lice, sheep keds, and even spiders, and hosts including humans, cats, dogs, rats, cattle, foxes, and bats (Breitschwerdt 2017). Importantly, at least 17 species have now been implicated in human disease (Breitschwerdt 2017; K. E. Mullins et al. 2015).
Disease presentation varies among Bartonella species. B. bacilliformis infection, thought to be transmitted by sandflies, is limited to the Andes mountain region of South America at elevations of 500-3,600 meters above sea level and causes Carrion's disease, which includes an acute hemolytic syndrome (Oroya fever) as well as a chronic condition (verruga peruana) (Jacomo, Kelly, and Raoult 2002;C. Gomes and Ruiz 2018;Clemente et al. 2012). B. quintana, transmitted by human body lice, and B. henselae, transmitted by fleas have worldwide distribution and are classically known to cause trench fever, known for its hallmark symptom, a relapsing fever, and the self-limited infection cat scratch disease, which presents as a fever with swollen lymph nodes, respectively (Jacomo, Kelly, and Raoult 2002;Karem, Paddock, and Regnery 2000). More recently, Bartonella species have been implicated in a wide range of disease manifestations from asymptomatic infection to mild fever and maculopapular rash to severe disease such as endocarditis, hallucinations, and even death (Breitschwerdt 2017;Cheslock and Embers 2019).
Due to the fastidious nature and lack of awareness of Bartonella species as human pathogens, the true extent of human exposure and disease associated with this genus is not fully understood (Cheslock and Embers 2019;Jacomo, Kelly, and Raoult 2002). Worldwide distribution and the increasing number of Bartonella species implicated in human disease suggests that Bartonella infections likely play a larger role in human disease than Peru-Serum samples from 48 patients were collected during a 2003 outbreak of B. bacilliformis in Huancambamba Province. Samples were collected from patients with suspected B. bacilliforms infections. Thirteen of the suspected cases were confirmed by PCR, culture, and/or identification of B. bacilliforms in blood smears. Samples were screened to assess the extent of the exposure to B. bacilliformis and to assess the ability of the assay to detect anti-B. bacilliformis IgG antibodies. Further, 206 serum samples collected between 2008 and 2015 from individuals with undifferentiated fever in the Amazonas, Junín, Cusco, and Loreto Regions of Peru were screened for the presence of IgG antibodies to Bartonella species (Santiago et al. 2015). Sample intergrity was assessed by were storred at -70 0 C until use.

Vector-Surveys
DNA extracted from fleas, lice, sandflies, and from previous vector studies was used for the detection of Bartonella species.
In Cambodia, 140 flea pools (2 to 25 fleas/pool) were collected from November of 2015 through January of 2016 using combing/brushing technique from dogs, cats, rats and mice found in Rantanakiri and Stung Treng Provinces. Of those 14 pools of Ctenocephalides felis and Pulex irritans were collected from cats, 119 pools of C. felis, Echidnophaga gallinacea, P. irritans were collected from dogs, and 7 pools of Xenopsylla cheopis were collected from mice and rats. Fleas were preserved in 70% ethanol, stored in cooler box, and transferred to laboratory. Fleas were morphologically identified using standard taxonomic keys and pooled by species and location (Menier and Beaucornu, 1930) . Fleas were briefly washed with distilled water and the DNA were extracted using DNeasy blood and tissue kit (Qiagen, Germany) following manufacturer's instruction. Sample intergrity was assesed by Nano-Drop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and were storred at -70 0 C until testing.
In Laos, fleas, ticks and lice were collected in March of 2018 from dogs in Vientiane City and pooled based on species and host, resulting in 22 flea pools, 106 tick pools, and 3 louse pools. Flea and louse pools contained 2-20 individual C. felis fleas or Heterodoxus spiniger lice, while 210 Rhipacephalus sanguineus ticks were pooled. DNA was extracted and stored as described in Nguyen et al, 2020. (Nguyen et al. 2020 In Peru, sandflies were collected in the Ancash region in 1999 and the Cajamarca region in 2012-2013 resulting in 99 and 100 pools of 2-10 sandflies/pool based on morphological detection, respectively (Romero 2004;Zorrilla et al. 2021). Sandflies were stored in 70% ethanol at -70 0 C until DNA was extracted. DNA were extracted using DNeasy blood and tissue kit (Qiagen, Germany) following a protocol modified from the manufacturer's instructions (Depaquit et al. 2005). DNA was stored at -70 0 C and sample intergity was assessed by Nano-Drop spectrophotometer.
Bartonella species were detected using a Bartonella genus-specific qPCR assay, BartA, as described in Flores Mendoza et al, 2021 which targets the ftsZ gene (Flores-Mendoza et al. 2021). In brief, each reaction contained 0.7 μM of the forward and reverse primers and 0.4 μM of the probe. Three microliters of template DNA was added to each reaction. The cycler parameters included an initial incubation at 50°C for 2 minutes followed by an initial denaturation at 95°C for 2 minutes, then 45 cycles of denaturation at 95 °C for 15 seconds and annealing/elongation at 60 °C for 30 seconds. Bartonalla ancashensis genomic DNA was used as the positive control.

Statistical analysis
Data managment and statistical analysis was performed in Microsoft (Redmond, WA) Excel. Minimum infection rate (MIR) was caculated: ([number of positive pools / total specimens tested] x 1000).

Vector-Surveys
The minimum infection rate of the flea pools from dogs, mice and rats found in the Ratanakiri and Strung Treng provinces of Cambodia was found to be 13.37% (0-39). Surprisingly, no flea pools collected from cats in the same provinces were positive. Flea species positive for Bartonella species DNA included C. felis, P. irritans, and X. cheopis.

Discussion and Conclusions
Bartonella species are increasingly common and recent studies have highlighted the widespread nature of Bartonella species (both human pathogens and non-human pathogens) infecting vectors such as fleas, ticks, lice and small mammal reservoir hosts worldwide (Cheslock and Embers 2019). The molecular detection of Bartonella species in flea pools collected from dogs, mice, and rats from Ratanakiri and Stung Treng provinces of Cambodia in this study is to our knowledge the first report of Bartonella species in fleas from Cambodia. However, a few studies of rodents and small mammals from Cambodia have found molecular evidence of Bartonella infection ranging from 7-10% (Duong et al. 2013;Jiyipong et al. 2015). Our data from fleas coupled with the data supporting Bartonella species present in small mammals and rodents provides evidence of the widespread distribution of Bartonella species (pathogenic and/or non-pathogenic to humans) in Cambodia.
However, surprisingly, flea and louse pools collected from the dogs in Vientiane Capital, Laos were negative for Bartonella species DNA. Previous studies in Laos found evidence of Bartonella species in fleas (including fleas collected from dogs) with positivity rates ranging from 3.3-33%, but these studies were outside of Vientiane Capital and there is evidence that levels of infection in rodents are significantly lower in human settlements (Varagnol et al. 2009;Jiyipong et al. 2015;Kernif et al. 2012;Calvani et al. 2020). Further, Bartonella species DNA was not found in ticks from Vientiane Capital, which is consistent with a previous study in which Bartonella species DNA was not detected in ticks collected from dogs and cats in Northern Laos (Kernif et al. 2012). While studies in the USA, Italy, Korea, and Austria have found evidence of Bartonella species in ticks ranging from 0.1 to 34.5%, ticks are not implicated as a major vector for human transmission of Bartonella species (Telford III and Wormser 2010;Persichetti et al. 2016;Satta et al. 2011;Kim et al. 2005;Adelson et al. 2004;Chang et al. 2001;Müller et al. 2016). An unfortunate and major limitation of our molecular surveys was the inability to provide sequence confirmation or speciation of Bartonella in these vectors and determine if the Bartonella species present in the animal reservoir or vector are pathogenic to humans. Nevertheless, this molecular evidence of Bartonella species in Southeast Asia supports previous reports and provides additional context for the results of the accompanying serosurveys and overall human exposure to Bartonella in Asia and worldwide.
While there are many studies of vectors and reservoir hosts, fewer studies investigate the prevalence of human exposure to (past or recent), or infection with, Bartonella species. Human Bartonella infections are described in case reports, most commonly endocarditis and cat-scratch disease, analysis of hospital records and insurance data, and from serosurveys with positivity ranges from 1% for healthy individuals in Sweden to 83% for sanitary workers in Spain (Daly et al. 1993;K. E. Mullins et al. 2015;Oteo et al. 2017;Müller et al. 2016;Im et al. 2018;Kwon et al. 2017;Portillo et al. 2020;Chomel et al. 2006;Bhengsri et al. 2011;MARUYAMA et al. 2000;Comer et al. 1996;Sun et al. 2010;Edouard et al. 2015;Fenollar, Sire, and Raoult 2005;FOURNIER et al. 2001;Kristin E. Mullins et al. 2013;Nawrocki et al. 2020;Alonso et al. 2020;Sandoval et al. 2020;Allizond et al. 2019). In this study, recent or previous Bartonellaexposure was evaluated in febrile patients in Cambodia, Ghana, Laos, and Peru. To our knowledge these studies represent the first human serosurveys in Cambodia and Laos, and only the second study in Ghana, where the first focused on bat-associated Bartonella (Mannerings et al. 2016). In this study the overall seroprevelance in the countries surveyed was consistent with previous reports (which vary widely) and ranged from 22-57%, although results from our serosurvey could overestimate prevalence of antibodies in the general (or healthy) populations due to the possibility that some patients with undifferentiated fevers were actively infected with Bartonella. Interestingly, Laos had the lowest seroprevelance, which could be accounted for with the lack of molecular detection of Bartonella species in fleas in Vientiane Capital, and possibly why the first reports of B. henselae endocarditis were only reported in 2014 (Rattanavong et al. 2014). Higher seroprevelance was seen in Cambodia and Ghana at 56% and 57%, respectively. Differences in seroprevelance likely correspond with lifestyle, occupation, and vector positivity rates, and contact with vectors and reservoir hosts, among other risk factors. This study was not able to elucidate possible risk factors for Bartonella exposure due to limited data on the individuals for which serum was available. However, this additional evidence of human exposure to Bartonella from places with limited previous research provides support that larger studies to determine the impact of Bartonella on human heath are needed especially in Cambodia, Laos, and Ghana. Further, this evidence argues for more aggressive evaluation of Bartonella species as a potential causes of the undifferentiated febrile illness in these locations especially upon report of an exposure to a known vector.
Finally, exposure to Bartonella species in Peru is unique as the Bartonella species of major importance, B. bacilliformis, which causes both acute and chronic infections, is found only in the Andes Mountain range, and thought to be vectored by sandflies(C. Gomes and Ruiz 2018;Clemente et al. 2012 This indicates that smear, PCR, and culture may currently miss a large portion of infected individuals, or that many of these individuals had prior exposure, but not active infection. Given the cross-reactivity observed among known Bartonella species in serological assays, including the FOCUS IFA used in this study, the study participants may have been exposed to Bartonella species that have yet to identified or characterized, or even those that do not cause illness in humans.
This highlights the need for more in depth investigations into the Bartonella species in humans, animals, and vectors in countries such as those included in this study, but also in countries and regions where knowledge of these agents is lacking.
Further, Bartonella species DNA was not identified in any sandfly pools from the endemic areas of Ancash and Cajamarca. Interestingly, all previous studies have found low levels of B. bacilliformis infection of sandflies ranging from 0.5-2.8 % (Ulloa et al. 2018;Ellis et al. 1999). While L. verrucarum is considered a known vector of B. Bacilliformis, other sandfly species have been implicated as possible vectors including L. maranosesis and L. robusta, both of which were included in this study, however the mechanisms by which B. bacilliformis is transmitted by sandflies is still lacking (Ulloa et al. 2018;Ellis et al. 1999;Garcia-Quintanilla et al. 2019;Clemente et al. 2012). The absence of molecular detection of Bartonella species in sandflies from endemic areas provides more insight into the epidemic nature of B. bacilliformis, and possibly B. ancashensis, for which a vector has yet to be identified (Kristin E Mullins et al. 2017;K. E. Mullins et al. 2015). Outbreaks of B. bacilliformis could be driven by asymptomatic carriers and immediate transmission between persons via sandflies. Studies have shown that outbreaks occur within families and members of a household are 2.6 times more likely to contract B. bacilliformis than neighbors (Cláudia Gomes et al. 2016). Additionally, asymptomatic carriage of B. bacilliformis is known to occur, with up to 40% of individuals in a post-exposure setting positive by qPCR (Chamberlin et al. 2002;Cláudia Gomes et al. 2016). The seroprevelance rate of 38% for B. bacilliformis in individuals with undifferentiated fever endemic areas is consistent with previous studies of post-exposure or endemic asymptomatic populations which found seropositivity rates between 19-67% (Cláudia Gomes et al. 2016;Chamberlin et al. 2002;Knobloch et al. 1985;Chamberlin et al. 2000). Our data likely includes both exposure to B. bacilliformis, as seen during the outbreak, as well as other Bartonella species including those which are not known to be human pathogens. Nevertheless, this data combined with previous reports indicates exposure to Bartonella in Peru is of continuing importance.
This study combined with previous data indicates that human exposure to Bartonella species is extensive and that Bartonella species may be the cause of undifferentiated fever in a subset of the individuals from these studies. These data taken along with previous work indicate that Bartonella species are likely an under-represented as cause of human disease around the world. Mullins et al. Page 14