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

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.

An Innovative Method for Rapid Identification and Detection of Vibrio alginolyticus in Different Infection Models

“…The mouse experiment was approved and supervised by Beijing Institute of Radiation Medicine Experiment Committee (2012-0131). The way of preparation of infected mouse model followed the methods described by Khajanchi et al (2011) , Angkasekwinai et al (2014) , and Liu et al (2014 , 2015 ).…”

Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples

“…These results are in accordance with Angkasekwinai et al . [ 21 ], who reported a detection limit of 1 and 10 copies/μL in a loop-mediated isothermal amplification when the detection limit was determined using bacterial genomic DNA alone or in the presence of human plasma respectively. This sensitivity might allow diagnosing the acute cases of Carrion’s disease, in which the mean percentage of infected RBCs is 61% (ranging from 2 to 100%) [ 22 ].…”

“…It is important to remark that in the last years 2 more sensitive PCR techniques have been described in the literature: qPCR [ 13 ] in which 24.6% of DBS samples are positive, as well as a loop-mediated isothermal amplification [ 21 ] that achieves good results on analysing Lutzomyia samples. However, qPCR requires the expertise of trained personnel and is more expensive and difficult to be implemented.…”

Carrion's Disease: the Sound of Silence

“…On the other hand, a qPCR targeting the NADH dehydrogenase gamma subunit gene (nuoG) was found to be sensitive and specific enough to detect diverse Bartonella species, maintaining minimal cross-reactivity to mammalian host and arthropod vector organisms (94). Angkasekwinai et al (275) described a loop-mediated isothermal amplification with pap31 as the target gene. This is a simple method to detect the presence of B. bacilliformis DNA within 1 h and requires less specialized equipment.…”