Abstract
BackgroundCarrion' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis. Methods and FindingsThe sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis.ConclusionsThe LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector.
Highlights
Carrion’s disease, caused by Bartonella bacilliformis remains truly neglected due to its focal geographical nature, occurring in small rural communities of inter-Andean valleys between altitudes of 500–200 meters above sea level in Peru, Columbia and Ecuador [1]
The loop-mediated isothermal amplification (LAMP) assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and to identify B. bacilliformis in the sand fly vector
As the period of asymptomatic bacteremia may vary and last for up to 15 months, these individuals can presumably serve as a reservoir for the bacteria, leading to potential transmission by the putative sand fly vector, Lutzomyia verrucarum, to either populations living in endemic areas or travelers visiting such regions [3]
Summary
Carrion’s disease, caused by Bartonella bacilliformis remains truly neglected due to its focal geographical nature, occurring in small rural communities of inter-Andean valleys between altitudes of 500–200 meters above sea level in Peru, Columbia and Ecuador [1]. L. verrucarum have been found throughout the western half of Peru between 1500 and 3200 meters above sea level in Occidental and Interandean valleys of the Andes mountain [9] This species was presumed to be the primary vector of B. bacilliformis, based on the evidence derived from the presence of the flies in the areas in which the disease occurred [8]. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap gene to detect B. bacilliformis
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