The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

Abstract

Background: Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis.

Methodology/principal findings: A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21%-85.63%) and 100% specificity (95% CI: 93.08%-100%).

Conclusions/significance: We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas.

Fig 1. Lamp primer set targeting the selected sequence (GenBank Accession No. AJ223838) for ribosomal intergenic spacer S. haematobium DNA region amplification.

(A) The location of the LAMP primers within the selected sequence is shown. Arrows indicate the direction of extension. (B). Sequence of LAMP primers: F3, forward outer primer; B3, reverse outer primer; FIP, forward inner primer (comprising F1c and F2 sequences); BIP, reverse inner primer (comprising B1c and B2 sequences); LF (loop forward primer); LB (loop backward primer).

Fig 2. PCR verification, detection limit and specificity using outer primers F3 and B3.

(A) PCR verification of expected 199 bp target length amplicon. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lane Sh, S. haematobium DNA (1 ng); lane N, negative control (no DNA template). (B) Detection limit of PCR. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lane Sh: S. haematobium DNA (1 ng); lanes 10⁻¹–10⁻⁹: 10-fold serially dilutions of S. haematobium DNA; lane N, negative control (no DNA template). (C) Specificity of PCR. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sh, Sm, Sj, Sb, Fh, Dd, Hd, Cd, Ll, Bp, As, Sv, Ts, Tt, Eg, Gi, Eh, Cp, Po, Pv, Pm, S. haematobium, S. mansoni, S. japonicum, S. bovis, Fasciola hepatica, Dicrocoelium dendriticum, Hymenolepis diminuta, Calicophoron daubneyi, Loa loa, Brugia pahangi, Anisakis simplex, Strongyloides venezuelensis, Trichinella spiralis, Taenia taeniformis, Echinococcus granulosus, Giardia intestinalis, Entamoeba histolytica, Cryptosporidium parvum, Plasmodium ovale, P. vivax and P. malariae DNA samples (1 ng/each), respectively; lane N, negative control (no DNA template).

Fig 3. Setting up LAMP assay.

(A) LAMP amplification results obtained at different incubation times (30, 50 and 60 min) tested in a heating block by the addition of SYBR Green I (up) or by visualization on agarose gel (down). Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes 30, 50, 60, amplification results of S. haematobium DNA (1 ng) for 30, 50 and 60 minutes of incubation time, respectively. (B) Specificity of the LAMP assay for S. haematobium. A ladder of multiple bands of different sizes could be only observed in S. haematobium DNA sample. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sh, Sm, Sj, Sb, Fh, Dd, Hd, Cd, Ll, Bp, As, Sv, Ts, Tt, Eg, Gi, Eh, Cp, Po, Pv and Pm, S. haematobium, S. mansoni, S. japonicum, S. bovis, Fasciola hepatica, Dicrocoelium dendriticum, Hymenolepis diminuta, Calicophoron daubneyi, Loa loa, Brugia pahangi, Anisakis simplex, Strongyloides venezuelensis, Trichinella spiralis, Taenia taeniformis, Echinococcus granulosus, Giardia intestinalis, Entamoeba histolytica, Cryptosporidium parvum, Plasmodium ovale, P. vivax and P. malariae DNA samples (1 ng/each), respectively; lane N, negative control (no DNA template). (C) Sensitivity assessment performed with LAMP at 63°C for 50 min using serial dilutions of S. haematobium genomic DNA. Lane M: 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sh: genomic DNA from S. haematobium (1 ng); lanes 10⁻¹–10⁻⁹: 10-fold serially dilutions; lane N: negative controls (no DNA template).

Fig 4. Sensitivity of the LAMP assay in simulated human urine samples artificially contaminated with DNA from S. haematobium.

(A) Sensitivity assessment of LAMP when performing the DNA extraction with the i-genomic Urine DNA Extraction Mini Kit (Intron Biotechnology, UK) from serial dilutions of S. haematobium genomic DNA. (B) Sensitivity assessment of LAMP when performing the DNA extraction with a simple heating method from serial dilutions of S. haematobium genomic DNA. Lanes M: 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sh: genomic DNA from S. haematobium (1 ng); lanes 10⁻¹–10⁻¹¹: 10-fold serially dilutions; lanes N: negative controls (no DNA template).

Fig 5. Examination of aliquots of whole urine from S. haematobium-positive patients´ urine samples by LAMP.

Figure shows the LAMP results (up, by color change; down, by agarose electrophoresis) when using aliquots of 100 μL of whole urine to obtain DNA as template by using (A) the i-genomic Urine DNA Extraction Mini Kit (Intron Biotechnology, UK); (B) the heating NaOH-SDS method and (C) the rapid heating method. Lanes M: 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sh: genomic DNA from S. haematobium (1 ng); lanes 1–18: S. haematobium-positive samples; lanes N: negative controls (no DNA template).

Fig 6. Examination of aliquots of supernatants from S. haematobium-positive patients´ urine samples by LAMP.

Figure shows the LAMP results (up, by color change; down, by agarose electrophoresis) when using aliquots of 100 μL of supernatants to obtain DNA as template by using (A) the i-genomic Urine DNA Extraction Mini Kit (Intron Biotechnology, UK); (B) the heating NaOH-SDS method and (C) the rapid heating method. Lanes M: 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sh: genomic DNA from S. haematobium (1 ng); lanes 1–18: S. haematobium-positive samples; lanes N: negative controls (no DNA template).

Fig 7. Examination of aliquots of urinary sediment (pellets) from S. haematobium-positive patients´ urine samples by LAMP.

Figure shows the LAMP results (up, by color change; down, by agarose electrophoresis) when using aliquots of 100 μL of pellets to obtain DNA as template by using (A) the i-genomic Urine DNA Extraction Mini Kit (Intron Biotechnology, UK); (B) the heating NaOH-SDS method and (C) the rapid heating method-the rapid-heat LAMPellet method-. Lanes M: 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sh: genomic DNA from S. haematobium (1 ng); lanes 1–18: S. haematobium-positive samples; lanes N: negative controls (no DNA template).