Strong-LAMP Assay Based on a Strongyloides spp.-Derived Partial Sequence in the 18S rRNA as Potential Biomarker for Strongyloidiasis Diagnosis in Human Urine Samples

Abstract

Human strongyloidiasis a soil-transmitted infection caused by Strongyloides stercoralis is one of the most neglected amongst the so-called Neglected Tropical Diseases (NTDs). S. stercoralis is a nematode, which is distributed worldwide; it has been estimated that it could affect millions of people, mainly in tropical and subtropical endemic regions. The difficulties of diagnosis lead to infection rates being underreported. Asymptomatic patients have chronic infections that can lead to severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. Strongyloidiasis can easily be misdiagnosed because conventional faecal-based techniques lack of sensitivity for the morphological identification of infective larvae in faeces. None of the currently used molecular methods have used urine samples as an alternative to faecal samples for diagnosing strongyloidiasis. This study was thus aimed at comparing, for the first time, the use of a new loop-mediated isothermal amplification (LAMP) molecular assay (Strong-LAMP) to traditional methods on patients' urine samples. Twenty-four urine samples were taken from patients included in a study involving two Spanish hospitals for strongyloidiasis screening using parasitological and serological tests. Strongyloides larvae were found in 11 patients' faecal samples, thereby ascertaining that they had the disease. Other patients had high antibody titres but no larvae were found in their faeces. All urine samples were analysed by PCR and Strong-LAMP assay. No amplification occurred when using PCR. Strong-LAMP led to detecting S. stercoralis DNA in urine samples from patients having previously confirmed strongyloidiasis by parasitological tests and/or a suspicion of being infected by serological ones. The Strong-LAMP assay is a useful molecular tool for research regarding strongyloidiasis in human urine samples. After further validation, the Strong-LAMP assay could also be used for complementary and effective diagnosis of strongyloidiasis in a clinical setting.

Figure 1

TD-PCR analysis of patients' urine samples. (a) PCR for verifying F3 and B3 primer functioning for amplifying the 214 bp fragment. (b) Samples from the Hospital de Poniente (El Ejido, Almería). (c) Samples from the Hospital Clínic de Barcelona. M: molecular weight marker (DNA Ladder 100 bp PLUS BLUE); C+: Strongyloides venezuelensis DNA (positive control); C−: ultrapure water, no DNA (negative control).

Figure 2

Strong-LAMP analysis of urine samples from patients attending Hospital Clínic de Barcelona (Barcelona). (a) Colorimetric results from adding SYBR Green I. (b) Agarose gel electrophoresis results. (c) Serological (IVD index, OD values) and agar plate culture results. M: molecular weight marker (DNA Ladder 100 bp PLUS BLUE); C+: Strongyloides venezuelensis DNA (positive control); C-: ultrapure water, no DNA (negative control).

Figure 3

Strong-LAMP analysis of urine samples from patients attending Hospital de Poniente (El Ejido, Almería). (a) Colorimetric results by adding SYBR Green I. (b) Agarose gel electrophoresis results. (c) Serological (IVD index, OD values), Ritchie technique and agar plate culture results. M: molecular weight marker (DNA Ladder 100 bp PLUS BLUE); C+: Strongyloides venezuelensis DNA (positive control); C-: ultrapure water, no DNA (negative control).

Figure 4

Agreement between Strong-LAMP, IVD detection techniques, and coproparasitological analysis in the studied samples with strongyloidiasis.