Development and evaluation of real-time loop-mediated isothermal amplification assay for rapid detection of cystic echinococcosis

Abstract

BackgroundCystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)-complex, is a neglected parasitic disease of public health importance. The disease is endemic in many African and Mediterranean countries including the Sudan. The objective of the present study was to develop and evaluate a real-time loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of CE in humans and domestic live stock in Sudan.MethodsA set of six LAMP primers, designed from the mitochondrial NADH-1 gene of EG cattle strain of genotype 5 (G5), was used as a target for LAMP assay. The assay was performed at a constant temperature (63 °C), with a real-time follow-up using a LightCycler and fluorochrome dye. Following amplification cycles in a simple water bath, LAMP products were observed for color change by naked eye and were visualized under UV light source using agarose gel electrophoresis.ResultsThe real-time LAMP assay identified a variety of hydatid cysts strains recovered in the Sudan, including Echinococcus canadenses (G6) and Echinococcus ortleppi (G5). Real-time LAMP positive results were detected by the presence of an amplification curve, whereas negative results were indicated by absence of fluorescence detection. Positive LAMP results appeared as a bluish-colored reaction as observed by naked eye, whereas negative LAMP results were observed as purple-colored reaction. The sensitivity studies indicated that the LAMP assay detected as little as a 10 fg of parasite DNA. There was 100 % agreement between results of the LAMP assay and our previously described nested PCR when testing 10-fold serial dilution of DNA extracted from EG-complex hydatid cyst. However, there was no cross-reactivity with other parasites including cysticercus bovis, Fasciola gigantica, and Schistosoma bovis and nucleic acid free samples.ConclusionThe developed LAMP assay would be expected to prove highly significant in epidemiological surveys of CE in developing countries or areas of resource-poor settings for both ease of use and cost.

Detection of helminths by loop-mediated isothermal amplification assay: a review of updated technology and future outlook

“…The impressive progress currently made in the LAMP assay for helminths includes trematodes of Clonorchis sinensis [12, 26, 60], Opisthorchis viverrini [14, 61, 62], Amphimerus spp. [63, 64], Paragonimus westermani [15], Fasciola hepatica [65–67], F. gigantica [65], Schistosoma japonicum [16, 27, 68–70], S. mansoni [13, 71–77], S. haematobium [51, 71, 72, 76]; nematodes of Necator americanus [78, 79], Ascaris lumbricoides [17, 79] , Trichuris trichiura [79], Toxocara canis [80] and T. cati [81], Strongyloides stercoralis [52, 82], Onchocerca volvulus [83–86], Wuchereria bancrofti [86, 87], Brugia malayi [86, 88], B. tomori [88], Loa loa [89–91], Dirofilaria repens [92], Angiostrongylus cantonensis [93, 94], Trichinella spiralis [95, 96], Bursaphelenchus xylophilus [97], and Haemonchus contortus [98, 99]; cestodes of T. solium [44, 100–103] , T. saginata [44, 100–103], T. asiatica [44, 100–103], T. hydatigena [104], T. multiceps [104], T. pisiformis [104] and T. crassiceps [104], Echinococcus granulosus [104–106], E. multilocularis [104, 107], E. equinus [108], E. canadensis [108], E. felidi [108], E. ortleppi [108, 109] and E. shiquicus [104] , have been covered in this review for further insight into its adoption for clinical diagnosis, field surveys and surveillance of helminths. The sensitivity and specificity of...…”

Development of real-time PCR assay for simultaneous detection and genotyping of cystic echinococcosis in humans and livestock

“…Loop-mediated isothermal amplification (LAMP) assays have been developed and proved highly sensitive and specific for diagnosis of echinococcosis in the canine definitive hosts [33][34][35]. However, LAMP assays were described for limited applications and were not used on a wide scale.…”

Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections

“…This involves using immunological methods such as enzyme-linked immunosorbent assay for coproantigen detection (coproELISA) and PCR. However, coproELISA can give false positives due to crossed reactions with other Taenia infections (including T. hydatigena or T. ovis) [39] whereas conventional PCR and quantitative real-time PCR (qRT-PCR) require sophisticated, expensive equipment, as well as infrastructure which cannot always be found in developing countries [41].…”

“…Standardised LAMP test sensitivity has been significant so far, even surpassing that of conventional PCR. It should be stressed that such tests can be made in remote rural areas using only a water bath or heat block and that the results can be interpreted by the naked eye [41]. Molecular diagnosis is useful for screening in the field and detecting DNA from eggs in environmental samples as an important step in identifying high-risk contaminated areas and establishing/ascertaining the routes for human infection, thereby enabling control programme progress to be determined [37].…”

“…Regarding the nad1 gene, authors have used B3 and BIP common primers, each one having an additional nucleotide at the 5 or 3 extreme of the sequence, whilst all the primers used for nad5 were different. Differences regarding the units in which the authors expressed analytical sensitivity limit results hampered comparison; nevertheless, it can be stated that greater sensitivity could be obtained (10 fg) with the primers used by Ahmed et al [41] for amplifying the nad1 gene; depending on the Echinococcus species being analysed, this sensitivity can be matched also when amplifying a cox1 fragment (10-100 fg) with the primers selected by Avila et al [42].…”