Detection of Schistosoma mekongi DNA in Human Stool and Intermediate Host Snail Neotricula aperta via Loop-Mediated Isothermal Amplification Assay in Lao PDR

Abstract

Schistosomiasis mekongi infection represents a public health concern in Laos and Cambodia. While both countries have made significant progress in disease control over the past few decades, eradication has not yet been achieved. Recently, several studies reported the application of loop-mediated isothermal amplification (LAMP) for detecting Schistosoma DNA in low-transmission settings. The objective of this study was to develop a LAMP assay for Schistosoma mekongi using a simple DNA extraction method. In particular, we evaluated the utility of the LAMP assay for detecting S. mekongi DNA in human stool and snail samples in endemic areas in Laos. We then used the LAMP assay results to develop a risk map for monitoring schistosomiasis mekongi and preventing epidemics. A total of 272 stool samples were collected from villagers on Khon Island in the southern part of Laos in 2016. DNA for LAMP assays was extracted via the hot-alkaline method. Following the Kato-Katz method, we determined that 0.4% (1/272) of the stool samples were positive for S. mekongi eggs, as opposed to 2.9% (8/272) for S. mekongi DNA based on the LAMP assays. Snail samples (n = 11,762) were annually collected along the riverside of Khon Island from 2016 to 2018. DNA was extracted from pooled snails as per the hot-alkaline method. The LAMP assay indicated that the prevalence of S. mekongi in snails was 0.26% in 2016, 0.08% in 2017, and less than 0.03% in 2018. Based on the LAMP assay results, a risk map for schistosomiasis with kernel density estimation was created, and the distribution of positive individuals and snails was consistent. In a subsequent survey of residents, schistosomiasis prevalence among villagers with latrines at home was lower than that among villagers without latrines. This is the first study to develop and evaluate a LAMP assay for S. mekongi detection in stools and snails. Our findings indicate that the LAMP assay is an effective method for monitoring pathogen prevalence and creating risk maps for schistosomiasis.

Keywords: LAMP; Laos; Schistosoma mekongi; risk map.

Figure 1

Study areas of Don Khon (Khon Island), Champasak Province, Laos. A map of Khon Island is also presented. Red stars show the locations where stool samples were collected from the study participants in 2016. Light blue circles show sampling sites of the host snail N. aperta from 2016 to 2018. Latitude and longitude are shown in parentheses. Background satellite image: WorldView2 © [2018] Maxar Technologies.

Figure 2

Sensitivity and specificity of LAMP assay that targets the ITS1 gene of S. mekongi. The LAMP assay was able to detect 1 pg of S. mekongi DNA, but neither S. mansoni nor S. japonicum DNA was detected. The left panel shows the LAMP results under daylight. The right panel shows LAMP results obtained under UV light. Fluorescence (light green) was confirmed in the reaction tubes containing S. mekongi DNA (10 pg and 1 pg).

Figure 3

Mapping of LAMP-positive residents (schistosomiasis mekongi patients) and host snails, yielding a heat map of S. mekongi infection risk. (A) All houses in Khon Island were plotted as circles on the map. The snail sampling sites were also plotted as triangles (in 2016–2018). Locations of LAMP-positive residents and host snails are shown in red on the map (LAMP data in 2016). (B) A heat map was made using the kernel density estimation based on the data of LAMP-positive residents (red) and host snails (blue). Background satellite image: WorldView2 © [2018] Maxar Technologies.