Diagnosis of Carrion's disease by direct blood PCR in thin blood smear negative samples.
ABSTRACT: Bartonella bacilliformis is the etiologic agent of Carrion's disease. This disease has two well established phases, the most relevant being the so called Oroya Fever, in which B. bacilliformis infect the erythrocytes resulting in severe anemia and transient immunosuppression, with a high lethality in the absence of adequate antibiotic treatment. The presence of B. bacilliformis was studied in 113 blood samples suspected of Carrion's disease based on clinical criteria, despite the absence of a positive thin blood smear, by two different PCR techniques (using Bartonella-specific and universal 16S rRNA gene primers), and by bacterial culture. The specific 16S rRNA gene primers revealed the presence of 21 B. bacilliformis and 1 Bartonella elizabethae, while universal primers showed both the presence of 3 coinfections in which a concomitant pathogen was detected plus Bartonella, in addition to the presence of infections by other microorganisms such as Agrobacterium or Bacillus firmus. These data support the need to implement molecular tools to diagnose Carrion's disease.
Project description: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.
Project description:Carrion's disease (CD) is a neglected biphasic vector-borne illness related to Bartonella bacilliformis. It is found in the Andean valleys and is transmitted mainly by members of the Lutzomyia genus but also by blood transfusions and from mother to child. The acute phase, Oroya fever, presents severe anemia and fever. The lethality is high in the absence of adequate treatment, despite the organism being susceptible to most antibiotics. Partial immunity is developed after infection by B. bacilliformis, resulting in high numbers of asymptomatic carriers. Following infection there is the chronic phase, Peruvian warts, involving abnormal proliferation of the endothelial cells. Despite potentially being eradicable, CD has been expanded due to human migration and geographical expansion of the vector. Moreover, in vitro studies have demonstrated the risk of the development of antimicrobial resistance. These findings, together with the description of new Bartonella species producing CD-like infections, the presence of undescribed potential vectors in new areas, the lack of adequate diagnostic tools and knowledge of the immunology and bacterial pathogenesis of CD, and poor international visibility, have led to the risk of increasing the potential expansion of resistant strains which will challenge current treatment schemes as well as the possible appearance of CD in areas where it is not endemic.
Project description:Here we present the complete genome sequence of Bartonella ancashensis strain 20.00, isolated from the blood of a Peruvian patient with verruga peruana, known as Carrion's disease. Bartonella ancashensis is a Gram-negative bacillus, phylogenetically most similar to Bartonella bacilliformis, the causative agent of Oroya fever and verruga peruana.
Project description:Carrion's disease is typically biphasic with acute febrile illness characterized by bacteremia and severe hemolytic anemia (Oroya fever), followed by benign, chronic cutaneous lesions (verruga peruana). The causative agent, Bartonella bacilliformis, is endemic in specific regions of Peru and Ecuador. We describe atypical infection in an expatriate patient who presented with acute splenomegaly and anemia 3 years after visiting Ecuador. Initial serology and PCR of the patient's blood and serum were negative for Bartonella henselae, Bartonella quintana, and B. bacilliformis. Histology of splenic biopsy was suggestive of bacillary angiomatosis, but immunohistochemistry ruled out B. henselae and B. quintana. Bacilli (isolate EC-01) were subsequently cultured from the patient's blood and analyzed using multilocus sequence typing, protein gel electrophoresis with Western blotting, and an immunofluorescence assay (IFA) against a panel of sera from patients with Oroya fever in Peru. The EC-01 nucleotide sequences (gltA and internal transcribed spacer) and protein band banding pattern were most similar to a subset of B. bacilliformis isolates from the region of Caraz, Ancash, in Peru, where B. bacilliformis is endemic. By IFA, the patient's serum reacted strongly to two out of the three Peruvian B. bacilliformis isolates tested, and EC-01 antigen reacted with 13/20 Oroya fever sera. Bacilliary angiomatosis-like lesions were also detected in the spleen of the patient, who was inapparently infected with B. bacilliformis and who presumably acquired infection in a region of Ecuador where B. bacilliformis was not thought to be endemic. This study suggests that the range of B. bacilliformis may be expanding from areas of endemicity in Ecuador and that infection may present as atypical clinical disease.
Project description:Carrion's disease is caused by infection with the alpha-proteobacterium Bartonella bacilliformis. Distribution of the disease is considered coincident with the distribution of its known vector, the sand fly Lutzomyia verrucarum. Recent epidemics of B. bacilliformis infections associated with atypical symptomatology in nonendemic regions have raised questions regarding the historic and present distribution of this bacterium and the scope of disease that infection causes. Phylogenetic relationships and genomic diversity of 18 B. bacilliformis isolates (10 isolates from a region where Carrion's disease is epidemic, Cuzco, Peru, and 8 isolates from a region where Carrion's disease is endemic, Caraz, Peru) were assessed using genomic data generated by infrequent restriction site PCR and gene sequence analysis of the flagellin gltA and ialB genes. A population genetic analysis of the genomic diversity suggests that what was once considered an epidemic region of Peru did not result from the recent introduction of B. bacilliformis.
Project description:Bartonella bacilliformis is the etiological agent of human bartonellosis, which is highly endemic to Peru. Here, we report the first genome that was sequenced and analyzed from an isolate of B. bacilliformis strain INS, which originally was isolated from the blood of an infected patient with an acute phase of Carrion's disease from Jaén-Cajamarca, Peru.
Project description: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 ?-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.
Project description:Analysis of immune responses in Bartonella bacilliformis carriers are needed to understand acquisition of immunity to Carrion's disease and may allow identifying biomarkers associated with bacterial infection and disease phases. Serum samples from 144 healthy subjects from 5 villages in the North of Peru collected in 2014 were analyzed. Four villages had a Carrion's disease outbreak in 2013, and the other is a traditionally endemic area. Thirty cytokines, chemokines and growth factors were determined in sera by fluorescent bead-based quantitative suspension array technology, and analyzed in relation to available data on bacteremia quantified by RT-PCR, and IgM and IgG levels measured by ELISA against B. bacilliformis lysates. The presence of bacteremia was associated with low concentrations of HGF (p = 0.005), IL-15 (p = 0.002), IL-6 (p = 0.05), IP-10 (p = 0.008), MIG (p = 0.03) and MIP-1? (p = 0.03). In multi-marker analysis, the same and further TH1-related and pro-inflammatory biomarkers were inversely associated with infection, whereas angiogenic chemokines and IL-10 were positively associated. Only EGF and eotaxin showed a moderate positive correlation with bacteremia. IgM seropositivity, which reflects a recent acute infection, was associated with lower levels of eotaxin (p = 0.05), IL-6 (p = 0.001), and VEGF (p = 0.03). Only GM-CSF and IL-10 concentrations were positively associated with higher levels of IgM (p = 0.01 and p = 0.007). Additionally, IgG seropositivity and levels were associated with high levels of angiogenic markers VEGF (p = 0.047) and eotaxin (p = 0.006), respectively. Our findings suggest that B. bacilliformis infection causes immunosuppression, led in part by overproduction of IL-10. This immunosuppression probably contributes to the chronicity of asymptomatic infections favoring B. bacilliformis persistence in the host, allowing the subsequent transmission to the vector. In addition, angiogenic markers associated with bacteremia and IgG levels may be related to the induction of endothelial cell proliferation in cutaneous lesions during chronic infections, being possible candidate biomarkers of asymptomatic infections.
Project description:BACKGROUND:Bartonella bacilliformis is the etiological agent of Carrion's disease, a neglected tropical poverty-linked illness. This infection is endemic of Andean regions and it is estimated that approximately 1.7 million of South Americans are at risk. This bacterium is a fastidious slow growing microorganism, which is difficult and cumbersome to isolate from clinical sources, thereby hindering the availability of phylogenetic relationship of clinical samples. The aim of this study was to perform Multi Locus Sequence Typing of B. bacilliformis directly in blood from patients diagnosed with Oroya fever during an outbreak in Northern Peru. METHODOLOGY/PRINCIPAL FINDINGS:DNA extracted among blood samples from patients diagnosed with Oroya's fever were analyzed with MLST, with the amplification of 7 genetic loci (ftsZ, flaA, ribC, rnpB, rpoB, bvrR and groEL) and a phylogenetic analysis of the different Sequence Types (ST) was performed. A total of 4 different ST were identified. The most frequently found was ST1 present in 66% of samples. Additionally, two samples presented a new allelic profile, belonging to new STs (ST 9 and ST 10), which were closely related to ST1. CONCLUSIONS/SIGNIFICANCE:The present data demonstrate that B. bacilliformis MLST studies may be possible directly from blood samples, being a promising approach for epidemiological studies. During the outbreak the STs of B. bacilliformis were found to be heterogeneous, albeit closely related, probably reflecting the evolution from a common ancestor colonizing the area. Additional studies including new samples and areas are needed, in order to obtain better knowledge of phylogenetic scenario B. bacilliformis.
Project description:Bartonella bacilliformis is the biological agent of Carrion's disease, a vector-borne, life-threatening human bartonellosis restricted to South America. Here, we report the complete genome sequence of B. bacilliformis KC584 (ATCC 35686). Although it is commonly used as a reference strain, to date, its complete genome has not been published.