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Image_1_Next step towards point-of-care molecular diagnosis of female genital schistosomiasis (FGS): evaluation of an instrument-free LAMP procedure.png

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posted on 2024-05-13, 09:46 authored by Kim J. M. van Bergen, Eric A.T. Brienen, Bodo S. Randrianasolo, Charles E. Ramarokoto, Peter Leutscher, Eyrun F. Kjetland, Angela van Diepen, Floris Dekker, Vittorio Saggiomo, Aldrik H. Velders, Lisette van Lieshout

Detection of Schistosoma spp. DNA in gynaecological samples by quantitative real-time polymerase chain reaction (qPCR) is considered to be the reference diagnostic test for female genital schistosomiasis (FGS). However, qPCR needs expensive laboratory procedures and highly trained technicians. Loop-mediated amplification (LAMP) is a more field-friendly isothermal procedure for the detection of parasite-specific DNA, but it still requires electrically powered equipment. Here, we validated a Schistosoma haematobium-specific Sh-LAMP procedure and tested a fully instrument-free isothermal amplification using a novel low-cost, and reusable Temperature-cup (T-cup) device. Specific primers were selected based on published assays, targeting the ribosomal intergenic spacer (IGS) region of S. haematobium. Technical validation of the IGS-Sh-LAMP was performed using 20 negative controls, including DNA extracts of soil-transmitted helminths and S. mansoni, and a 10-fold dilution series (100–10−3) of DNA extracted from a single S. haematobium egg (n=4). For clinical validation, the IGS-Sh-LAMP was tested on 125 DNA samples extracted from vaginal swabs of a previous FGS study in Madagascar. Results were compared with the quantification cycle value (Cq) of the standard ITS-2 targeting qPCR. Single S. haematobium egg DNA up to a 10–2 dilution and an ITS-2 Cq <35 tested positive in the IGS-Sh-LAMP. The specificity was found to be excellent (100%). In the clinical samples, IGS-Sh-LAMP showed comparable results with the qPCR, with 35.2% and 33.6% positives, respectively, and a concordance of 79.2% (99/125). Of the 12 false-negatives, 5 corresponded to the 7 qPCR positive samples with very low DNA levels (Cq ≥35). On the other hand, IGS-Sh-LAMP detected 14 additional cases that were not detected by qPCR. The T-cup IGS-Sh-LAMP performance was evaluated in a representative sub-selection (n=10) of IGS-Sh-LAMP positive clinical samples. The T-cup IGS-Sh-LAMP was found to be a very user-friendly method, but in different runs, it missed 1 to 4 of the 10 IGS-Sh-LAMP positive samples, specifically those with a low DNA load. Our results show that the IGS-Sh-LAMP is a suitable alternative to the ITS-2 qPCR for the diagnosis of FGS in gynaecological samples, with high potential for the T-cup as a fully instrument-free isothermal amplification device for point-of-care diagnosis in low-resource settings.

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