Loop-Mediated Isothermal Amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts

Abstract

BackgroundAlveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis.MethodsThe primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People’s Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs.ResultsThe E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge.ConclusionThe earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas.

Intestinal Carriage of Carbapenemase-Producing Organisms: Current Status of Surveillance Methods

“…These assays were able to detect all 48 tested isolates with either NDM or KPC, while endpoint PCR detected only 44. Other studies have found an improved performance of LAMP versus endpoint PCR for microbiological targets other than CPE but not versus real-time assays (180,181). Therefore, LAMP assays may have a useful role in detecting CPOs, but they are not the most sensitive assay for clinical microbiology laboratories that have access to other types of NAATs.…”

Echinococcosis: Advances in the 21st Century

“…As is discussed further below, molecular diagnosis, including loop-mediated isothermal amplification (LAMP), can be used as a first-line screen for Echinococcus spp. in the field (134)(135)(136)(137) and to detect Echinococcus sp. egg DNA in environmental samples as an important step for identifying high-risk contaminated areas and for defining the actual routes of human infection (138)(139)(140)(141)(142).…”

“…As indicated above, LAMP-based assays are useful as a first-line screen for Echinococcus spp. in the field (134)(135)(136)(137), and PCR methods allow combined identification of definitive host species and Echinococcus sp. infection status using feces collected in the field (260).…”

Novel approaches to the diagnosis of Strongyloides stercoralis infection

“…It is characterized by the use of six different primers specifically designed to recognize eight distinct regions on a target gene, with amplification only occurring if all primers bind and form a product [66]. In the last few years, parasitologists have adapted the LAMP approach for the detection of several parasites, including Echinococcus [67][68][69], Taenia [70][71][72], Loa loa [73,74], Opistorchis [75], Clonorchis [76], filariae [77], and Trichinella [78]. The method appears to be promising for S. stercoralis too [79], although it still needs to be validated in clinical practice [13].…”