Research Article: Rapid and Sensitive Detection of Bartonella bacilliformis in Experimentally Infected Sand Flies by Loop-Mediated Isothermal Amplification (LAMP) of the Pap31 Gene

Date Published: December 18, 2014

Publisher: Public Library of Science

Author(s): Nasikarn Angkasekwinai, Erin H. Atkins, Richard N. Johnson, John P. Grieco, Wei Mei Ching, Chien Chung Chao, Mathieu Picardeau. http://doi.org/10.1371/journal.pntd.0003342

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.

Partial Text: 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 disease typically presents with acute fever and devastating hemolysis known as Oroya fever, followed by asymptomatic bacteremia and chronic eruptive disease, called Verruga peruana [2]. 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]. The increase of human migration, habitat destruction, as well as adaptation of sand flies association with human, may also aid the spread of the potential vector [4]. With changes in weather patterns or host dynamics, sporadic and occasional epidemics of bartonellosis have occurred over a much larger area than previously described [5]–[8].

We reported here the development of the LAMP assay targeting the pap31 gene to detect B. bacilliformis. The limit of detection (LOD) for the LAMP assay ranged from 1 to 10 copies/µL, depending on the sample metrics, which was comparable to that of real-time PCR which ranged from 2 to 18 copies/µL. In addition, no cross-reaction was observed when testing a panel of other closely related bacteria. We also demonstrated the capability of the pap31 LAMP assay to identify B. bacilliformis in the sand fly vector. 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.

Source:

http://doi.org/10.1371/journal.pntd.0003342

 

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