Development and evaluation of a loop-mediated isothermal amplification (LAMP) diagnostic test for detection of whipworm, Trichuris trichiura, in faecal samples

Abstract

Whipworm infection or trichuriasis caused by Trichuris trichiura is of major public health concern in sub-Saharan Africa, particularly among pre-school and school-going children. It is among the neglected tropical diseases targeted for elimination through mass drug administration (MDA). One of the outcomes of MDA is a rapid decline in levels of infection intensity, making it difficult to monitor effectiveness of control measures using the conventional Kato–Katz procedure, which relies on the microscopic detection of parasite ova in faecal samples. In the present study, a loop-mediated isothermal amplification (LAMP) test was developed for the detection of T. trichiura infection in faecal samples. LAMP technology offers greater sensitivity and specificity than the microscopy-based tests. A set of four specific primers targeting the internal transcribed spacer 2 region of the ribosomal DNA were designed using Primer Explorer software. DNA was extracted from faecal samples using the alkaline lysis method (HotSHOT) and the LAMP reaction performed at 63°C for 1 h. The amplicons were visualized by both gel electrophoresis and with the naked eye following staining with SYBR green dye. Sensitivity and specificity tests were determined using the standard Kato–Katz diagnostic procedure as a reference test. The developed LAMP assay reliably detected T. trichiura DNA in faecal samples, with a specificity and sensitivity of 88% and 77%, respectively. No cross-reactivity was observed with several common helminth parasites. The developed LAMP assay is an appropriate diagnostic method for the detection of T. trichiura DNA in human faecal samples due to its simplicity, low cost, high sensitivity and specificity.

Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples

“…The Copro-LAMPAc was able to detect 100 fg of gDNA from A. caninum adult worms, which is more sensitive than previously reported values for other PCR assays ( 33 – 35 , 66 ). This analytical sensitivity value is included in the femtograms range, similar to what has been obtained in other studies ( 38 , 49 , 74 ). In future studies, the A. caninum egg limit of detection could be determined.…”

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

“…No positive PCR results were obtained when using the same primers on urine samples [173]. Concerning T. trichiura, the set of primers which amplified the ITS-2 region designed by Ngari et al [172] was able to detect very low levels of infection, having greater sensitivity than the Kato-Katz technique. Even though the detection limit obtained by Rashwan et al [174] after designing primers amplifying the β-tubulin region was not better (1 pg), the advantage of this protocol lies in its ability to detect T. trichiura, A. lumbricoides and N. americanus eggs.…”

“…Even though the detection limit obtained by Rashwan et al [174] after designing primers amplifying the β-tubulin region was not better (1 pg), the advantage of this protocol lies in its ability to detect T. trichiura, A. lumbricoides and N. americanus eggs. Even though the Kato-Katz technique's sensitivity and specificity are low and LAMP's detection limit is considered good, work is still needed on optimising protocols for improving the sensitivity and specificity obtained for T. trichiura (77%, sensitivity, 88% specificity [172]) and for A. lumbricoides (96.3% sensitivity, 61.5% specificity [176]).…”

LAMP in Neglected Tropical Diseases: A Focus on Parasites

“…2 In this category the parasite and the intermediate host are excluded. 3 Detection methods used were: SYBR Green I [ 28 , 35 , 36 , 37 , 44 , 45 , 47 , 55 , 57 , 60 , 61 , 62 , 63 , 64 , 66 , 68 , 72 , 74 , 80 , 81 , 83 , 87 , 90 , 92 , 93 , 97 , 98 , 99 ], electrophoresis [ 25 , 28 , 34 , 35 , 37 , 41 , 44 , 45 , 47 , 55 , 58 , 61 , 62 , 63 , 65 , 75 , 79 , 80 , 81 , 83 ,...…”