Cytokine Profile Response of Human Peripheral Blood Mononuclear Cells Stimulated by Bartonella bacilliformis.

Carrion's disease is a neglected endemic disease found in remote Andean areas. As an overlooked disease, knowledge of innate immune responses to Bartonella bacilliformis, the etiological agent, is scarce. This study aimed to evaluate the cytokine response to B. bacilliformis using in vitro human peripheral blood mononuclear cells (PBMCs) stimulations. PBMCs from naive adults were isolated by gradient centrifugation and cocultured with heat-inactivated (HI) B. bacilliformis at different incubation times (3, 6, 12, 24, and 36 h). Cytokines, chemokines, and growth factors were determined in culture supernatants by multiplex fluorescent bead-based quantitative suspension array technology. During the first 36 h, a proinflammatory response was observed, including tumor necrosis factor-α, interleukin (IL)-1α, IL-1ß, interferon-α2, and IL-6, followed by an anti-inflammatory response mainly related to IL-1RA. Moreover, high expression levels of chemokines IL-8, monocyte chemoattractant protein-1α, and macrophage inflammatory protein (MIP)-1ß were detected from 3 h poststimulation and MIP-1α was detected at 24 h. Some growth factors, mainly granulocyte macrophage colony-stimulating factor and granulocyte colony-stimulating factor, and in minor concentrations vascular endothelial growth factor, epidermal growth factor, and eotaxin, were also detected. Innate response to HI B. bacilliformis stimulation consists of a rapid and strong proinflammatory response characterized by a wide range of cytokines and chemokines followed by an anti-inflammatory response and increased specific growth factors.

Single-nucleotide polymorphisms in ialB, gltA and rpoB genes of Bartonella bacilliformis isolated from patients in endemic Peruvian regions.

Bartonella bacilliformis is a Gram-negative, aerobic bacterium and the known causal agent of Carrion's disease, still considered a neglected disease. There is limited information about the nucleotide sequences of this bacterium in international databases, and few studies have addressed the genetic diversity of B. bacilliformis. We analyzed a total of 20 isolates of B. bacilliformis from the Peruvian regions of Ancash and Cajamarca. Three genes (ialB, gltA, and rpoB) were sequenced in each isolate and nucleotide sequences retrieved from GenBank (16 B. bacilliformis genomes) were also included in the study. All this information was merged in order to obtain clearer evidence of the phylogenetic relationships of B. bacilliformis. In the phylogenetic analysis conducted with the concatenated markers, four isolates (B.b-1, B. b-3, B. b- 7, B.b-8) from the Ancash region were observed to form a subgroup different from B. bacilliformis type strain KC583, showing dissimilarity levels of 5.96% (ialB), 3.69% (gltA) and 3.04% (rpoB). Our results suggest that B. bacilliformis consists of two different subgroups. Future investigations are needed to establish the taxonomic status of these subgroups.

In-silico identification of linear B-cell epitopes in specific proteins of Bartonella bacilliformis for the serological diagnosis of Carrion's disease.

Carrion´s disease is caused by Bartonella bacilliformis, it is a Gram-negative pleomorphic bacterium. B. bacilliformis is transmitted by Lutzomyia verrucarum in endemic areas of the Peruvian Inter-Andean valleys. Additionally, the pathogenicity of B. bacilliformis involves an initial infection of erythrocytes and the further infection of endothelial cells, which mainly affects children and expectant women from extreme poverty rural areas. Therefore, the implementation of serological diagnostic methods and the development of candidate vaccines for the control of CD could be facilitated by the prediction of linear b-cell epitopes in specific proteins of B. bacilliformis by bioinformatics analysis. In this study, We used an in-silico analysis employing six web servers for the identification of epitopes in proteins of B. bacilliformis. The selection of B. bacilliformis-specific proteins and their analysis to identify epitopes allowed the selection of seven protein candidates that are expected to have high antigenic activity.

A system for transposon mutagenesis of Bartonella bacilliformis.

Bartonella bacilliformis is the etiologic agent of Carrión's disease in South America. Lack of a system for random mutagenesis has significantly hampered research on the pathogen's molecular biology. Here, we describe a transposon (Tn)-based mutagenesis strategy for B. bacilliformis using pSAM_Rl; a Tn-mariner delivery vector originally constructed for members of the Rhizobiaceae family. Following electroporation of the vector, five candidate mutant strains were selected based on aberrant colony morphologies, and four mutations confirmed and identified using arbitrarily-primed PCR coupled with Sanger sequencing. One mutant strain, 4B2, was found to have a disrupted flgI gene, encoding the P-ring component of the flagellar motor. We therefore investigated the flgI strain's motility phenotype in a novel motility medium and found that insertional mutagenesis produced a non-motile mutant. Taken as a whole, the results show that: 1) pSAM_R1 is a practical Tn delivery vector for B. bacilliformis, 2) the plasmid can be used to create random Tn mariner mutants, 3) arbitrarily-primed PCR coupled with Sanger sequencing is a rapid and simple method for identifying and locating mutations generated by this Tn, and 4) in silico-predicted mutant phenotypes can be verified in vitro following mutagenesis. This system of Tn mutagenesis and mutation identification provides a novel and straightforward approach to investigate the molecular biology of B. bacilliformis.

Cloning, expression and seroreactivity of the recombinant lipopolysaccharide assembly protein - D (LptD) from Bartonella bacilliformis. / Clonamiento, expresión y seroreactividad de la proteína recombinante de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis.

OBJECTIVE.: To evaluate in silico and at the serological level the antigenic potential of the recombinant extracellular domain of the lipopolysaccharide assembly protein - D (LptD) of Bartonella bacilliformis (dexr_LptD). MATERIALS AND METHODS.: Through in silico analysis, we selected a B. bacilliformis protein with antigenic and immunogenic potential. The selected protein gene was cloned into Escherichia coli TOP10 and expressed in Escherichia coli BL21 (DE3) pLysS. Recombinant protein was expressed using isopropyl-ß-D-1-thiogalactopyranoside (IPTG) and induction conditions were optimized. Finally, it was purified with Ni-IDA resin (His60 Ni Superflow) and a Western Blot assay was conducted. RESULTS.: In silico, the selected protein was LptD because it is located in the outer membrane and is antigenic and immunogenic. Optimized conditions for dexr_LptD induction were 0.5 mM IPTG, 16 hours, TB (Terrific Broth) medium, 3% (v/v) ethanol, 28 ºC, OD600: 1-1.5 and 200 rpm. Purification was carried out under denaturating conditions on a small scale and we obtained 2.6 µg/mL of partially purified dexr_LptD. The Western Blot assay showed a positive reaction between the sera from patients with Carrión's Disease and dexr_LptD, which shows the antigenicity of dexr_LptD. CONCLUSIONS.: The dexr_LptD shows antigenicity both in silico and at the serological level, these results are the basis for further studies on vaccine candidates against Carrion's Disease.

OBJETIVO.: Evaluar in silico y a nivel serológico el potencial antigénico del dominio extracelular recombinante de la proteína de ensamblaje de lipopolisacáridos - D (LptD) de Bartonella bacilliformis (dexr_LptD). MATERIALES Y MÉTODOS.: Mediante el análisis in silico se realizó la selección de una proteína de B. bacilliformis con potencial antigénico e inmunogénico. El gen de la proteína seleccionada se clonó en Escherichia coli TOP10 y se expresó en Escherichia coli BL21 (DE3) pLysS. La proteína recombinante fue expresada usando isopropil-ß-D-1-tiogalactopiranósido (IPTG) y se optimizaron las condiciones de inducción. Por último, se purificó con resina Ni-IDA (His60 Ni Superflow) y se realizó un ensayo de Western Blot. RESULTADOS.: In silico, la proteína seleccionada fue LptD por estar localizada en la membrana externa y ser antigénica e inmunogénica. Las condiciones optimizadas para la inducción del dexr_LptD fueron 0,5 mM IPTG, 16 h, medio TB (Terrific Broth), etanol al 3% (v/v), 28 ºC, OD600: 1-1,5 y 200 r.p.m. La purificación se realizó en condiciones denaturantes a pequeña escala y se obtuvo 2,6 µg/mL de dexr_LptD parcialmente purificada. El ensayo de Western Blot mostró una reacción positiva entre los sueros provenientes de pacientes con la enfermedad de Carrión y dexr_LptD, ello evidencia la antigenicidad del dexr_LptD. CONCLUSIONES.: El dexr_LptD muestra antigenicidad in silico y a nivel serológico, estos resultados son base para posteriores estudios sobre candidatos vacunales contra la enfermedad de Carrión.

The Passenger Domain of Bartonella bacilliformis BafA Promotes Endothelial Cell Angiogenesis via the VEGF Receptor Signaling Pathway.

Bartonella bacilliformis is a Gram-negative bacterial pathogen that provokes pathological angiogenesis and causes Carrion's disease, a neglected tropical disease restricted to South America. Little is known about how B. bacilliformis facilitates vasoproliferation resulting in hemangioma in the skin in verruga peruana, the chronic phase of Carrion's disease. Here, we demonstrate that B. bacilliformis extracellularly secrets a passenger domain of the autotransporter BafA exhibiting proangiogenic activity. The B. bacilliformis-derived BafA passenger domain (BafABba) increased the number of human umbilical endothelial cells (HUVECs) and promoted tube-like morphogenesis. Neutralizing antibody against BafABba detected the BafA derivatives from the culture supernatant of B. bacilliformis and inhibited the infection-mediated hyperproliferation of HUVECs. Moreover, stimulation with BafABba promoted phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and extracellular-signal-regulated kinase 1/2 in HUVECs. Suppression of VEGFR2 by anti-VEGFR2 antibody or RNA interference reduced the sensitivity of cells to BafABba. In addition, surface plasmon resonance analysis confirmed that BafABba directly interacts with VEGFR2 with lower affinity than VEGF or Bartonella henselae-derived BafA. These findings indicate that BafABba acts as a VEGFR2 agonist analogous to the previously identified B. henselae- and Bartonella quintana-derived BafA proteins despite the low sequence similarity. The identification of a proangiogenic factor produced by B. bacilliformis that directly stimulates endothelial cells provides an important insight into the pathophysiology of verruga peruana. IMPORTANCE Bartonella bacilliformis causes life-threatening bacteremia or dermal eruption known as Carrion's disease in South America. During infection, B. bacilliformis promotes endothelial cell proliferation and the angiogenic process, but the underlying molecular mechanism has not been well understood. We show that B. bacilliformis induces vasoproliferation and angiogenesis by producing the proangiogenic autotransporter BafA. As the cellular/molecular basis for angiogenesis, BafA stimulates the signaling pathway of vascular endothelial growth factor receptor 2 (VEGFR2). Identification of functional BafA protein from B. bacilliformis in addition to B. henselae and B. quintana, the causes of cat scratch disease and trench fever, raises the possibility that BafA is a common virulence factor for human-pathogenic Bartonella.

JMM Profile: Bartonella bacilliformis: a forgotten killer.

Bartonella bacilliformis causes Carrión's disease, an infectious disease present in rural Andean areas of Peru and Ecuador. The disease has an acute and a chronic phase called Oroya fever and Peruvian wart, respectively. Oroya fever is potentially fatal if treated inadequately. Female Lutzomyia verrucarum, a phlebotomine sand fly endemic to South America, is the major vector. B. bacilliformis exhibits high susceptibility levels to a variety of antibacterial agents. B. bacilliformis is difficult to culture. Most endemic areas are remote with fragile health systems and poor communication. Thus, the true burden of the disease is difficult to ascertain.

Comparison of sand fly trapping approaches for vector surveillance of Leishmania and Bartonella species in ecologically distinct, endemic regions of Peru.

BACKGROUND: In Peru, the information regarding sand fly vectors of leishmaniasis and bartonellosis in the Amazon region is limited. In this study, we carried out sand fly collections in Peruvian lowland and highland jungle areas using different trap type configurations and screened them for Leishmania and Bartonella DNA. METHODOLOGY/PRINCIPAL FINDINGS: Phlebotomine sand flies were collected in Peruvian Amazon jungle and inter Andean regions using CDC light trap, UV and color LED traps, Mosquito Magnet trap, BG Sentinel trap, and a Shannon trap placed outside the houses. Leishmania spp. screening was performed by kDNA PCR and confirmed by a nested cytochrome B gene (cytB) PCR. Bartonella spp. screening was performed by ITS PCR and confirmed by citrate synthase gene (gltA). The PCR amplicons were sequenced to identify Leishmania and Bartonella species. UV and Blue LED traps collected the highest average number of sand flies per hour in low jungle; UV, Mosquito Magnet and Shannon traps in high jungle; and Mosquito Magnet in inter Andean region. Leishmania guyanensis in Lutzomyia carrerai carrerai and L. naiffi in Lu. hirsuta hirsuta were identified based on cytB sequencing. Bartonella spp. related to Bartonella bacilliformis in Lu. whitmani, Lu. nevesi, Lu. hirsuta hirsuta and Lu. sherlocki, and a Bartonella sp. related to Candidatus B. rondoniensis in Lu. nevesi and Lu. maranonensis were identified based on gltA gene sequencing. CONCLUSIONS/SIGNIFICANCE: UV, Blue LED, Mosquito Magnet and Shannon traps were more efficient than the BG-Sentinel, Green, and Red LED traps. This is the first report of L. naiffi and of two genotypes of Bartonella spp. related to B. bacilliformis and Candidatus B. rondoniensis infecting sand fly species from the Amazon region in Peru.

Identification of immunodominant Bartonella bacilliformis proteins: a combined in-silico and serology approach.

BACKGROUND: Bartonella bacilliformis is the aetiological agent of Carrión's disease, a biphasic and highly lethal illness formerly restricted to the South American Andes that is now spreading to adjacent areas. Reliable serodiagnostic approaches and vaccines are urgently needed. In this study, we aimed to identify immunodominant proteins of B bacilliformis and to establish novel and reliable serodiagnostic tools. METHODS: We used a reverse vaccinology approach in combination with an analysis of heterologous genomic expression libraries to identify immunodominant proteins, on the basis of the genome sequences of B bacilliformis strains KC583 and KC584. Antigens were screened with serum samples collected from Peruvian patients with B bacilliformis infections and from German healthy blood donors without history of travel to South America. We further analysed immunoreactive proteins of B bacilliformis with immunoblotting and line blots. We used selected target proteins to develop a diagnostic ELISA. To assess the performance of this ELISA, we did receiver operating characteristic analyses to assess the area under the curve, cutoff values, sensitivities, and specificities with 95% CIs. FINDINGS: We used serum samples obtained between Dec 23, 1990, and May 5, 2018, from 26 Peruvian patients with B bacilliformis infections and serum samples taken between Aug 28 and Aug 31, 2020, from 96 healthy German blood donors. 21 potentially immunodominant proteins were identified and recombinantly expressed, and their reactivity was assessed with immunoblotting and line blots. Of these 21 antigens, 14 were found to be immunoreactive. By using serum samples of Peruvian patients with Carrión's disease and of healthy German blood donors, we identified three antigens (porin B, autotransporter E, and hypothetical protein B) as suitable immunodominant antigens, and we applied them in a diagnostic ELISA using two different antigen combinations (porin B plus autotransporter E and porin B plus autotransporter E plus hypothetical protein B). For the combination of porin B and autotransporter E, with optical density measured at 450 nm (OD450) cutoff value of 0·29, sensitivity was 80·8% (95% CI 60·7-93·5) and specificity was 94·8% (88·3-98·3) for all Peruvian patient samples. For a combination of porin B, autotransporter E, and hypothetical protein B, with an OD450 cutoff of 0·34, sensitivity was 76·9% (56·4-91·0) and specificity was 93·8% (86·9-97·7) for all Peruvian patient samples. INTERPRETATION: This novel ELISA could represent a useful serodiagnostic tool for future epidemiological studies of B bacilliformis in endemic areas. Additionally, the immunodominant antigens we have identified could provide a first basis for future vaccine development to prevent the highly lethal Carrión's disease. FUNDING: DRUID (Novel Drug Targets against Poverty-Related and Neglected Tropical Infectious Diseases) Initiative and Robert Koch Institute. TRANSLATIONS: For the Spanish and Quechua translations of the abstract see Supplementary Materials section.

Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host.

Bartonella bacilliformis, the etiological agent of Carrión's disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión's disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9 (BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.

Subtractive proteomics and immunoinformatics approaches to explore Bartonella bacilliformis proteome (virulence factors) to design B and T cell multi-epitope subunit vaccine.

Bartonella bacilliformis a gram-negative facultative aerobe responsible for the Carrion's disease widely distributed in Ecuador, Peru, and Colombia with a high mortality rate when no specific treatment is received. B bacilliformis is transmitted by Sand fly (Lutzomyia verrucarum) to healthy individuals. Immunoinformatic and subtractive proteomics approaches were employed in this study to prioritize the best candidates for vaccine designing. These approaches resulted in five vaccine candidates, flagellar biosynthetic protein (Uniprot ID: A1UTU1), heme exporter protein C (UniProt ID: A1UU82), Cytochrome c-type biogenesis protein (Uniprot ID: A1URZ7), Hemin ABC transporter (Uniprot ID: A1US20) and Phosphatidate cytidylyltransferase (Uniprot ID: A1USE3). The mentioned proteins are antigenic and essential for pathogen survival. A range of immune-informatics tools was applied for the prediction of B and T cell epitopes for the vaccine candidate proteins. In-silico vaccine was constructed using carefully evaluated epitopes and consequently modeled for docking with human Toll-like receptor 4. TLR-4 agonist 50S ribosomal protein L7/L12 (UniproKB ID; P9WHE3) was linked to the vaccine as an adjuvant to boost immune response towards the vaccine. For stability evaluation of the vaccine-TLR-4 docked complex, MD simulations were performed. The final vaccine was back-translated and cloned in Eschericia coli to attain the maximal expression of the vaccine protein. The maximal expression was ensured, and the CAI score of 0.96 was reported. The current vaccine requires future experimental validation to confirm its effectiveness. The vaccine developed will be helpful to protect against B bacilliformis associated infections.

Prioritisation of potential drug targets against Bartonella bacilliformis by an integrative in-silico approach.

BACKGROUND Carrion's disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion's disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.

In silico analysis of Pap31 from Bartonella bacilliformis and other Bartonella spp.

Pap31 is an outer membrane protein of Bartonella bacilliformis which is considered to be a potential antigenic candidate for the development of diagnostic tools. The present study aimed to compare Pap31 from B. bacilliformis with that of other Bartonella spp. The results showed the presence of at least 5 different B. bacilliformis Pap31 alleles, with the strain Ver097 being the most divergent (89.7% of identity with the reference strain KC583). The most significant finding was the presence of a variable number (1 to 3) of 6 amino acid tandem repeats (GTEGGG) in the different B. bacilliformis Pap31 alleles, with no similar structure in other established Bartonella spp., except for Bartonella ancashensis, another Bartonella spp. isolated from chronic cases of Carrion's disease. In both B. bacilliformis and B. ancashensis this repetitive region was coincident with the most predicted immunogenic region of the protein. In other microorganisms, the presence of amino acid tandem repeats has been related to the development of poorly functional antibodies. The findings of this study also suggest a utility of Pap31 amino acid tandem repeats as potential contributors to the immune evasion of Carrion's disease-related Bartonella spp. and the establishment of asymptomatic B. bacilliformis / B. ancashensis infections.

Human vascular endothelial cells express epithelial growth factor in response to infection by Bartonella bacilliformis.

Bartonella are Gram-negative bacterial pathogens that trigger pathological angiogenesis during infection of humans. Bartonella bacilliformis (Bb) is a neglected tropical agent endemic to South America, where it causes Carrión's disease. Little is known about Bb's virulence determinants or how the pathogen elicits hyperproliferation of the vasculature, culminating in Peruvian warts (verruga peruana) of the skin. In this study, we determined that active infection of human umbilical vein endothelial cells (HUVECs) by live Bb induced host cell secretion of epidermal growth factor (EGF) using ELISA. Killed bacteria or lysates of various Bb strains did not cause EGF production, suggesting that an active infection was necessary for the response. Bb also caused hyperproliferation of infected HUVECs, and the mitogenic response could be inhibited by the EGF-receptor (EGFR) inhibitor, AG1478. Bb strains engineered to overexpress recombinant GroEL, evoked greater EGF production and hyperproliferation of HUVECs compared to control strains. Conditioned (spent) media from cultured HUVECs that had been previously infected by Bb were found to be mitogenic for naïve HUVECs, and the response could be inhibited by EGFR blocking with AG1478. Bb cells and cell lysates stimulated HUVEC migration and capillary-like tube formation in transmigration and Matrigel assays, respectively. To our knowledge, this is the first demonstration of EGF production by Bb-infected endothelial cells; an association that could contribute to hyperproliferation of the vascular bed during bartonellosis.

The protein-protein interactions required for assembly of the Tn3 resolution synapse.

The site-specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer-dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis ("Bart"). Tn3 and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific "R" interface involving resolvase NTDs at all three dimer-binding sites in res. Synapses containing mixtures of wild-type Tn3 and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimer-dimer interaction required for catalysis.

Dubious presence of Bartonella bacilliformis in ticks from Madre de Dios, Peru.

Bartonella bacilliformis has recently been described in Amblyomma scalpturatum, Amblyomma ovale and Rhipicephalus microplus collected from wild animals in the Peruvian region of Madre de Dios. In this communication, I will discuss the results of a recent study by del Valle-Mendoza et al. together with the B. bacilliformis epidemiology. Following my argumentation, I consider the presence of this microorganism in the above ticks improbable.

Carrion's disease: more than a neglected disease.

Infections with Bartonella bacilliformis result in Carrion's disease in humans. In the first phase of infection, the pathogen causes a hemolytic fever ("Oroya fever") with case-fatality rates as high as ~90% in untreated patients, followed by a chronical phase resulting in angiogenic skin lesions ("verruga peruana"). Bartonella bacilliformis is endemic to South American Andean valleys and is transmitted via sand flies (Lutzomyia spp.). Humans are the only known reservoir for this old disease and therefore no animal infection model is available. In the present review, we provide the current knowledge on B. bacilliformis and its pathogenicity factors, vectors, possible unknown reservoirs, established and potential infection models and immunological aspects of the disease.

Molecular detection and clinical characteristics of Bartonella bacilliformis, Leptospira spp., and Rickettsia spp. in the Southeastern Peruvian Amazon basin.

BACKGROUND: Acute febrile illness (AFI) represent a significant health challenge in the Peruvian Amazon basin population due to their diverse etiologies and the unavailability of specific on-site diagnostic methods, resulting in underreporting of cases. In Peru, one of the most endemic regions to dengue and leptospirosis is Madre de Dios, a region also endemic to emergent bacterial etiologic agents of AFI, such as bartonellosis and rickettsiosis, whose prevalence is usually underreported. We aimed to molecularly identify the presence of Leptospira spp., Bartonella bacilliformis, and Rickettsia spp. by Polymerase Chain Reaction in serum samples from patients with AFI from Puerto Maldonado-Madre de Dios in Peru. METHODS: Serum samples from patients with acute febrile illness were analyzed by real-time PCR for detecting the presence of Bartonella bacilliformis, Leptospira spp. and Rickettsia spp. RESULTS: Bartonella bacilliformis was the most prevalent bacteria identified in 21.6% (30/139) of the samples, followed by Leptospira spp. in 11.5% (16/139) and Rickettsia spp. in 6.5% (9/139) of the samples. No co-infections were observed between these bacteria. The most frequent symptoms associated with fever among all groups, were headaches, myalgias, and arthralgias. We found no statistically significant differences in the clinical presentation between patients infected with each bacterium. CONCLUSIONS: In a previous study, we shown the presence of dengue, chikungunya, Zika and oropouche virus. We were able to identify these pathogens in 29.5% of all the samples, with chikungunya and OROV as the most frequently found in 9.4 and 8.6% of all the samples, respectively. In this study we show that B. bacilliformis (21.6%), Leptospira spp. (11.5%) and Rickettsia spp. (6.5%) accounted for the main etiologies of AFI in samples from Puerto Maldonado-Madre de Dios, Perú. Our analysis of their clinical presentation, further shows the importance of implementing more sensitive and specific on-site diagnostic tools in the national surveillance programs.This study confirms that the un-specificity of signs and symptoms is not only associated with arboviral infections, but also with the clinical presentation of endemic bacterial infections.

Molecular Detection of Bartonella bacilliformis in Lutzomyia maranonensis in Cajamarca, Peru: A New Potential Vector of Carrion's Disease in Peru?

Carrion's disease is a neglected, vector-borne illness that affects Colombia, Ecuador, and especially Peru. The phlebotomine sand flies Lutzomyia verrucarum and Lutzomyia peruensis are the main illness vectors described, although other species may be implicated in endemic areas such as some northern Peruvian regions, in which Carrion's disease vector has not been established. The aim of this study was to evaluate the presence of Bartonella bacilliformis DNA in Lutzomyia maranonensis from Cajamarca, northern Peru. This sand fly has not been defined as a vector yet. Centers for Disease Control and Prevention light traps were used to collect adult phlebotomine sand flies from 2007 to 2008 in the Cajamarca department. Female specimens were identified using morphological keys and were grouped into pools of five sand flies, taking into account district and sampling site (intradomicile or peridomicile). DNA was extracted, and then conventional and real-time polymerase chain reaction (RT-PCR) were performed to detect B. bacilliformis and subsequently confirmed by sequencing. A total of 383 specimens of L. maranonensis species were analyzed. Two of 76 pools were positive for B. bacilliformis by sequencing; all positives pools were from Querocotillo district. In addition, Mesorhizobium spp. were identified in two pools of sand flies, which is an α-proteobacteria phylogenetically very close to B. bacilliformis. This study presents molecular evidence that suggests L. maranonensis is naturally infected by B. bacilliformis in the Cajamarca department. Further research should determine if L. maranonensis is a vector and could transmit B. bacilliformis.