Dataset Information

Evaluation of LAMP for detection of Shigella from stool samples in children.

ABSTRACT: Background: To assess the diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for the detection of Shigella from stool samples from children.

Methods: Consecutive stool samples from children aged <13?years old who presented with acute watery diarrhoea or dysentery to the Department of Paediatrics were collected and processed in the Department of Microbiology. All the stool samples were subjected to culture, conventional PCR and LAMP. Genomic sequencing was performed for samples that were positive by LAMP but negative by both culture and conventional PCR. The LAMP results were compared to those from culture and to a composite reference standard based on culture and conventional PCR.

Results: Amongst the 374 stool samples tested, 291 samples were positive by LAMP and 213 were positive by the composite reference standard. The sensitivity of LAMP was 100?% (98.3-100?%) and its specificity was 51.6?% (43.6-59.5?%) with a disease prevalence of 57?%. The sensitivity and specificity of LAMP improved to 99.3?% (94.2-100) and 98.2?% (94.5-99.9), respectively, using latent class analysis, while assuming that genomic sequencing has perfect specificity.

Discussion: The authors have standardized the LAMP procedure for direct application to clinical stool samples. LAMP is a sensitive and specific method for the diagnosis of Shigella from stool samples of children as compared to both culture and conventional PCR.

SUBMITTER: Raghavan R

PROVIDER: S-EPMC7717480 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Evaluation of LAMP for detection of <i>Shigella</i> from stool samples in children.

Raghavan Ramya R Wang Shouao S Dendukuri Nandini N Kar Sitanshu S SS Mahadevan Subramanian S Jagadisan Barath B Mandal Jharna J

Access microbiology 20200914 11

<h4>Background</h4>To assess the diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for the detection of <i>Shigella</i> from stool samples from children.<h4>Methods</h4>Consecutive stool samples from children aged <13 years old who presented with acute watery diarrhoea or dysentery to the Department of Paediatrics were collected and processed in the Department of Microbiology. All the stool samples were subjected to culture, conventional PCR and LAMP. Genomic sequencing was pe ...[more]

PMID: 33294772

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Project description:BACKGROUND:Trichuris trichiura (human whipworm) infects an estimated 477 million individuals worldwide. In addition to T. trichiura, other Trichuris species can cause an uncommon zoonosis and a number of human cases have been reported. The diagnosis of trichuriasis has relied traditionally on microscopy. Recently, there is an effort to use molecular diagnostic methods, mainly qPCR. LAMP technology could be an alternative for qPCR especially in low-income endemic areas. Trichuris muris, the causative agent of trichuriasis in mice, is of great importance as a model for human trichuriasis. Here, we evaluate the diagnostic utility of a new LAMP assay in an active experimental mouse trichuriasis in parallel with parasitological method by using stool and, for the first time, urine samples. METHODS:Stool and urine samples were collected from mice infected with eggs of T. muris. The dynamics of infection was determined by counting the number of eggs per gram of faeces. A LAMP based on the 18S rRNA gene from T. muris was designed. Sensitivity and specificity of LAMP was tested and compared with PCR. Stool and urine samples were analysed by both LAMP and PCR techniques. RESULTS:Trichuris muris eggs were detected for the first time in faeces 35 days post-infection. LAMP resulted specific and no cross-reactions were found when using 18 DNA samples from different parasites. The detection limit of the LAMP assay was 2 pg of T. muris DNA. When testing stool samples by LAMP we obtained positive results on day 35 p.i. and urine samples showed amplification results on day 20 p.i., i.e. 15 days before the onset of T. muris eggs in faeces. CONCLUSIONS:To the best of our knowledge, we report, for the first time, a novel LAMP assay (Whip-LAMP) for sensitive detection of T. muris DNA in both stool and urine samples in a well-established mice experimental infection model. Considering the advantages of urine in molecular diagnosis in comparison to stool samples, should make us consider the possibility of starting the use urine specimens in molecular diagnosis and for field-based studies of human trichuriasis where possible. Further studies with clinical samples are still needed.

Project description:BACKGROUND:Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. METHODOLOGY/PRINCIPAL FINDINGS:Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. CONCLUSIONS/SIGNIFICANCE:Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting.

Project description:BackgroundAmphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals. The diagnosis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes. No immunological or molecular methods have been developed to date. New diagnostic techniques for specific and sensitive detection of Amphimerus spp. DNA in clinical samples are needed.Methodology/principal findingsA LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region was designed. Amphimerus sp. DNA was obtained from adult worms recovered from animals and used to optimize the molecular assays. Conventional PCR was performed using outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target sequence. LAMP was optimized using different reaction mixtures and temperatures, and it was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using 44 human stool samples collected from localities where fluke is endemic. Twenty-five samples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing, PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all human stool samples included in our study, the diagnostic parameters for the sensitivity and specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%, respectively.Conclusions/significanceWe have developed and evaluated, for the first time, a specific and sensitive LAMP assay for detecting Amphimerus sp. in human stool samples. The procedure has been named LAMPhimerus method and has the potential to be adapted for field diagnosis and disease surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the applicability of this novel LAMP assay.

Dataset Information

Evaluation of LAMP for detection of Shigella from stool samples in children.

Publications

Evaluation of LAMP for detection of <i>Shigella</i> from stool samples in children.

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