Infectious diseases of poverty · 2026
Development and field evaluation of a multiplex qPCR assay for environmental DNA detection of Schistosoma mekongi and its intermediate snail host Neotricula aperta in the Mekong River Basin.
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He Jian, Andrus Peter S, Phonekeo Sengrloun, Yin Kun, Xiu Leshan, Lv Shan, Yang Kun, Sayasone Somphou, Zhou Xiao-Nong
Аннотация
BACKGROUND: Schistosoma mekongi remains an important cause of intestinal schistosomiasis in the lower Mekong River Basin. Sensitive environmental surveillance tools are needed to support transmission monitoring, particularly where conventional snail surveys are seasonally constrained. This study developed and evaluated a multiplex quantitative PCR (qPCR) assay for simultaneous detection of S. mekongi and its intermediate snail host, Neotricula aperta, in aquatic environmental DNA (eDNA) samples.
METHODS: Singleplex and multiplex qPCR assays targeting S. mekongi and N. aperta were compared to establish the optimal amplification system. Field validation was conducted in the Khong District, Champasak Province, Laos, during the dry season (May 2024) and wet season (August 2024) across 20 transmission sites. Method agreement between eDNA detection and malacological surveys was assessed using Cohen's kappa coefficient, and associations between N. aperta density and physicochemical variables were evaluated using Spearman's rank correlation.
RESULTS: The optimized multiplex qPCR assay targeting N. aperta (16S rRNA) and S. mekongi (18S rRNA) showed high sensitivity, with limits of detection of 5 and 6 copies/μl and limits of quantification of 40 and 46 copies/μl, respectively. In mesocosms, both targets were detected at an infected-snail density of 1 snail/L, with median Cq values of 35.23 for N. aperta and 26.54 for S. mekongi. During the dry-season surveys, 978 N. aperta were collected, with a mean density of 46.25 snails per site; wet-season surveys were not feasible. No schistosome infections were detected by crushing microscopy. Field eDNA analysis detected N. aperta but not S. mekongi, with N. aperta eDNA positivity being 90% in the dry season and 40% in the wet season. Agreement with malacological surveys was moderate (κ = 0.44; p = 0.01) and snail density was significantly associated with water temperature, pH, and dissolved oxygen.
CONCLUSIONS: This multiplex eDNA assay sensitively detected N. aperta in field water samples and may help overcome seasonal limitations of malacological surveys. Although S. mekongi was detected under laboratory conditions, it was not detected in field samples, consistent with the absence of infected snails. Further validation in active-transmission sites is required before reliable field detection of S. mekongi eDNA can be assumed.
METHODS: Singleplex and multiplex qPCR assays targeting S. mekongi and N. aperta were compared to establish the optimal amplification system. Field validation was conducted in the Khong District, Champasak Province, Laos, during the dry season (May 2024) and wet season (August 2024) across 20 transmission sites. Method agreement between eDNA detection and malacological surveys was assessed using Cohen's kappa coefficient, and associations between N. aperta density and physicochemical variables were evaluated using Spearman's rank correlation.
RESULTS: The optimized multiplex qPCR assay targeting N. aperta (16S rRNA) and S. mekongi (18S rRNA) showed high sensitivity, with limits of detection of 5 and 6 copies/μl and limits of quantification of 40 and 46 copies/μl, respectively. In mesocosms, both targets were detected at an infected-snail density of 1 snail/L, with median Cq values of 35.23 for N. aperta and 26.54 for S. mekongi. During the dry-season surveys, 978 N. aperta were collected, with a mean density of 46.25 snails per site; wet-season surveys were not feasible. No schistosome infections were detected by crushing microscopy. Field eDNA analysis detected N. aperta but not S. mekongi, with N. aperta eDNA positivity being 90% in the dry season and 40% in the wet season. Agreement with malacological surveys was moderate (κ = 0.44; p = 0.01) and snail density was significantly associated with water temperature, pH, and dissolved oxygen.
CONCLUSIONS: This multiplex eDNA assay sensitively detected N. aperta in field water samples and may help overcome seasonal limitations of malacological surveys. Although S. mekongi was detected under laboratory conditions, it was not detected in field samples, consistent with the absence of infected snails. Further validation in active-transmission sites is required before reliable field detection of S. mekongi eDNA can be assumed.