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

      Ruiz, J; Silva, W.; Tinoco, C.; Pons, Maria J; Pons, Maria J; Del Valle, Luis J.; Gomez, C.; Bazan, Jorge; Vargas, M.; Champin, D.; Mendoza, J. del Valle; juana.delvalle@upc.edu.pe (2014-07-17)
      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.
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    • Evaluation of three PCR schemes for detection of Bartonella bacilliformis in blood samples: sensitivity, specificity and applicability

      Universidad Peruana de Ciencias Aplicadas (UPC) (Elsevier B.V., 2015-03-24)
      Background: Bartonella bacilliformis is the etiological agent of Carrion’s disease, a neglected illness with a febrile lethal stage and a warty benign phase, being the human the only known reservoir. The diagnostic by microscopy in endemic areas is several times erroneous. Furthermore, the culture of this bacterium is time-consuming, being the diagnostic by PCR the easiest way to perform a correct diagnostic. The objective of this study was to evaluate the detection limit of three PCR schemes, designed to detect B.bacilliformis, both in blood and filter papers to test their potential use for transferring samples from endemic areas to reference centers. Moreover, the specificity was also observed as well as the applicability of the technique with clinical samples from different stages of the disease. Methods & Materials: Fragments of 16SrRNA and fla genes were amplified as well as the variable-intergenic region (its). The detection limit was determined by bacterial quantification with flow cytometry and performing dilutions (106cfu/ml-10cfu/ml) both in blood and filter papers. DNA was extracted and PCRs were performed. Specificity was tested by processing other bacteraemia microorganisms. Clinical samples, 12 from febrile patients, 13 from warty and 71 from healthy asymptomatic individuals living in endemic area(Mandinga-Cajamarca) were also processed. Results: The 16SrRNA PCR scheme showed the lower detection limit (5 cfu from blood and filter paper) being the PCR scheme chosen to be tested in clinical samples. All febrile patients’ samples were positive, whereas in warty individuals only 3(23%) faint bands were obtained. No amplification was obtained in samples from healthy people. Fainter bands were always obtained when PCRs were made of filter papers. All PCRs were specific for B.bacilliformis. Conclusion: The 16SrRNA PCR seems to be the best technique to detect feverish patients. However, the applicability to identify asymptomatic carriers was undetermined. Filter paper may be an alternative for easy transportation of samples but is need to consider the decreasing sensitivity of the results. It is critical to develop rapid, sensitive and specific technique capable of being applied in endemic rural areas, to avoid misdiagnosis and facilitate the detection of asymptomatic carriers that will allow progress towards the eradication of this disease.
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