A Case for Using Genomics and a Bioinformatics Pipeline to Develop Sensitive and Species-Specific PCR-Based Diagnostics for Soil-Transmitted Helminths

Grant, Jessica R.; Pilotte, Nils; Williams, Steven A.

doi:10.3389/fgene.2019.00883

Cited by 16 publications

(15 citation statements)

References 106 publications

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“…Prior STHcontrol programmes coupled with rapid economic development and improvements in infrastructure and policies are likely to have led to the elimination of STH transmission in Japan. This is the first study to use multi-parallel real-time PCR, a highly sensitive and specific molecular-based diagnostic method [35,53], for large-scale STH surveillance in Japan. Traditionally, the Kato's cellophane thick smear technique has been used for large-scale helminth surveys in Japan [54].…”

Section: Discussionmentioning

confidence: 99%

What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan

et al. 2020

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Background: Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs.Methods: Stool samples were collected from 682 preschool-and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura.Results: Among the 682 children, no positive samples were identified, and participants reported high standards of WASH. Conclusions:To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.

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Section: Discussionmentioning

confidence: 99%

What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan

et al. 2020

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“… 61 , 62 Ribosomal sequences and mitochondrial targets such as cytochrome oxidase are found in most eukaryotic cells making them attractive target choices. 62 In STH, a large portion of the genome is comprised of repetitive non-coding DNA elements, which are useful PCR targets for STH. 63 It should be noted that some types of repetitive DNA, such as simple short nucleotide repeats or sequence variation within the repeat, impact the suitability of repeat-based molecular assays.…”

Section: Molecular Methodsmentioning

confidence: 99%

Diagnostic Techniques for Soil-Transmitted Helminths – Recent Advances

Khurana¹,

Mewara²

2021

RRTM

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Soil-transmitted helminth (STH) infections (hookworms, Trichuris, Ascaris ) and Strongyloides spp. are associated with a substantial global burden and high morbidity. Sensitive and specific methods for diagnosis of these infections are essential for mapping the burden in communities, accurate assessment of infection levels, to guide interventions and monitoring the success of STH control programs. Despite considerable progress to control STH over several decades, we are still far from identifying a fully adequate diagnostic test. Conventional microscopy-based methods such as direct Kato–Katz smear or mounts after stool centrifugation/flotation-based concentration techniques have been the mainstay of diagnosis, especially in resource-poor countries where these infections abound. However, recently, these are being adapted to closed, easy to perform, digital formats, thereby improving the sensitivity as well as applicability in a remote, resource-limited setting. The use of image analysis systems to identify and quantify helminth eggs, with potential adaptation to smartphones, is also promising. Antibody detection tests have a limited role mostly in the case of Strongyloides hyperinfection. Coproantigen detection tests have been developed and used in veterinary practice for detection of STH, but these have not been evaluated for use in humans. More sensitive molecular diagnostics, including assays developed with new bioinformatic tools and techniques such as polymerase chain reaction (PCR), quantitative PCR (qPCR) and loop-mediated amplification assay, can help in the clear and precise assessment of STH burden during elimination phase and are of immense value for diagnosis in areas with low endemicity and in travelers to endemic regions. Moreover, the molecular techniques will help detect new species that may emerge. Sample preservation and efficient DNA extraction are critical and significantly affect the efficiency of molecular diagnostic tests. In addition to the diagnosis of clinical or asymptomatic infection in humans, detection of STH eggs in environmental samples is imperative to boost STH control efforts. Overall the diagnostic performance, cost-effectiveness, ease of performance, rapidity and in-field applicability of any test should be considered when choosing from the various diagnostic assays in areas with different endemicity, in addition to striving towards the development of novel technologies and optimization of existing methods.

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“…improvements to both analytical and clinical/field sensitivity and specificity of detection when compared with less prevalent, more conserved regions that have traditionally been employed [21,[24][25][26]. Under experimental conditions, such targets result in reduced cycle quantification (Cq) values for a given concentration of template DNA.…”

Section: Plos Onementioning

confidence: 99%

“…While PCR-based assays targeting the causative agents of LF and human malaria have existed for over two decades [19][20], recent advances in genome biology, next-generation sequencing (NGS), and bioinformatics are facilitating increasingly systematic approaches to assay design [21]. Through the computational analysis of whole genome sequencing data, highly repetitive, species-specific, tandemly repeated DNA elements can be readily and rapidly identified [22][23].…”

Section: Introductionmentioning

confidence: 99%

Selection and exploitation of prevalent, tandemly repeated genomic targets for improved real-time PCR-based detection of Wuchereria bancrofti and Plasmodium falciparum in mosquitoes

Zulch¹,

Pilotte²,

Grant³

et al. 2020

PLoS ONE

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Background Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both next-generation sequencing (NGS) technologies and bioinformaticsbased analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent. Methodology/Principal findings Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number. Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms. For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay. Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome's most abundant

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A Case for Using Genomics and a Bioinformatics Pipeline to Develop Sensitive and Species-Specific PCR-Based Diagnostics for Soil-Transmitted Helminths

Cited by 16 publications

References 106 publications

What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan

What does soil-transmitted helminth elimination look like? Results from a targeted molecular detection survey in Japan

Diagnostic Techniques for Soil-Transmitted Helminths – Recent Advances

Selection and exploitation of prevalent, tandemly repeated genomic targets for improved real-time PCR-based detection of Wuchereria bancrofti and Plasmodium falciparum in mosquitoes

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