Etiological and molecular diagnostic of Carrion’s disease in patients from Cajamarca - Perú

Abstract

Background: Bartonellagenus is a group of facultative intracellular pathogens that posses able to survive and proliferate inside of erythrocytes. Classified within this genus,Bartonella bacilliformisis of special relevance. This microorganism is the etiological agent of the so called Carrion’s Disease (Human bartonellosis). Additionally the presence of sub-clinical cases (asymptomatyc carriers) is of special interest, because acts as a reservoir of this illness. Carrión’s Disease is an endemic illnes in Perú, affecting in a special manner the north interandean valleys. However, the current in use diagnostic techniques (Giemsa Stain) possess low sensitivity and specificity, and due to the fact thatB. bacilliformispossess a low growth (weeks), bacterial cultures lacks of clinical utility. Thus suspictious cases frequently are not confirmed, and the real relevance of this illness remains underestimated. This work is addressed to the direct identification from blood samples ofBartonella baciliformisusing a conventional PCR. All patients were from the Cajamarca area being enrolled by the Epidemiological Surveillance program of DIRESA. Methods: The samples were processed at arriving to the laboratory, by molecular and microbiological techniques. Thus samples were cultured in Blood Columbia Agar (10%), in anaerobic conditions at 28 ◦C for a period of 2 months. Positive cultures were both Giemsa stained and identified by the amplification of a fragment the 16S rRNA gene. Genetic material was directly extracted from blood samples using the Kit High Pure (Roche diagnostic), and a fragment of 438 bp of the 16S rRNA gene was amplified withBartonellagenus specific primers. All positive PCR were sequenced (Macrogen-Korea). Results: A total of 134 blood samples were processed, from this 12 (8.9%) grown in blood agar, while in 18 (13.4%), including the aforementioned 12, the 16 s rRNA gene was amplified. In all cases the sequence analysis showed the presence ofB. bacilliformis Conclusion: Although microbiological culture is the gold standard in the identification ofBartonellaspp., this technique possess strong limitations due to the low growth of these microorganisms. However, the PCR is a rapid technique, possessing a high sensibility and specificity that may be used as routine diagnostic tool for the identification of Carrion’s Disease.