Research Article: Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia

Date Published: February 1, 2018

Publisher: Public Library of Science

Author(s): Bruce H. Noden, Jaclyn Martin, Yisel Carrillo, Justin L. Talley, Francisco M. Ochoa-Corona, Ulrike Gertrud Munderloh.

http://doi.org/10.1371/journal.pone.0192331

Abstract

The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results.

A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/μl (25μl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR.

To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries.

Partial Text

The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide as a principal cause of non-malarial febrile illness [1–4]. There are more than 25 bacteria, including species in the genera Anaplasma, Ehrlichia, Orientia, and Rickettsia, impacting human and animal health that are transmitted by vector-borne arthropods, mainly ticks or fleas [2]. While most are treatable with antibiotics, several are difficult to diagnose at low infection levels [5,6]. Reports continue to demonstrate that these tick- and flea-borne pathogens are impacting human and animal populations in low resource countries which lack technical and material capabilities. Often, these countries are not aware that these pathogens are circulating in their populations [1,3,7,8]. In recent years, increased funding and research focus has shifted to the development of point-of-care diagnostics that will detect the causative agents of neglected tropical diseases [6]. While some of the diagnostics show promise in the detection of vector-borne bacterial pathogens in their arthropod vectors [5], little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries.

Since its introduction in 2000, many studies have used some form of LAMP technology to detect vector-borne pathogens. The majority of the studies, however, developed LAMP assays to directly detect pathogen DNA in blood, sputum, feces, and skin of animals, including humans. By the end of 2016, only 2 LAMP studies detecting tick-transmitted bacterial pathogens had tested field-collected ticks during the development of their LAMP assays ((Borrelia burgdorferi [13] and Ehrlichia ruminantium [14]). No LAMP studies to date have screened fleas for rickettsial pathogens. This study introduces a functional SFGR-LAMP assay applicable as a surveillance tool to prompt initial screening of SFG and TRG rickettsia DNA in field-collected ticks and fleas.

 

Source:

http://doi.org/10.1371/journal.pone.0192331

 

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