<i>Ancylostoma ceylanicum</i> Hookworm in Myanmar Refugees, Thailand, 2012–2015

O’Connell, Elise M.; Mitchell, Tarissa; Papaiakovou, Marina; Pilotte, Nils; Lee, Dong Hoon; Weinberg, Michelle; Sakulrak, Potsawin; Tongsukh, Dilok; Oduro-Boateng, Georgiette; Harrison, Sarah M.; Williams, Steven A.; Stauffer, William M.; Nutman, Thomas B.

doi:10.3201/eid2408.180280

Cited by 16 publications

(17 citation statements)

References 42 publications

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“…A study by O'Connell et al on Myanmar refugees living in Thailand found a baseline prevalence of 5.4% for An. ceylanicum [44]. Hookworm species cannot be differentiated under the microscope, so it is likely that many studies have been including An.…”

Section: Diagnostic Sensitivity and Methodology Testingmentioning

confidence: 99%

The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration

et al. 2020

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Background: The most commonly used diagnostic tool for soil-transmitted helminths (STH) is the Kato-Katz (KK) thick smear technique. However, numerous studies have suggested that the sensitivity of KK can be problematic, especially in low prevalence and low intensity settings. An emerging alternative is quantitative polymerase chain reaction (qPCR). Methods: In this study, both KK and qPCR were conducted on stool samples from 648 participants in an STH epidemiology study conducted in the delta region of Myanmar in June 2016. Results: Prevalence of any STH was 20.68% by KK and 45.06% by qPCR. Prevalence of each individual STH was also higher by qPCR than KK, the biggest difference was for hookworm with an approximately 4-fold increase between the two diagnostic techniques. Prevalence of Ancylostoma ceylanicum, a parasite predominately found in dogs, was 4.63%, indicating that there is the possibility of zoonotic transmission in the study setting. In individuals with moderate to high intensity infections there is evidence for a linear relationship between eggs per gram (EPG) of faeces, derived from KK, and DNA copy number, derived from qPCR which is particularly strong for Ascaris lumbricoides. Conclusions: The use of qPCR in low prevalence settings is important to accurately assess the epidemiological situation and plan control strategies for the 'end game'. However, more work is required to accurately assess STH intensity from qPCR results and to reduce the cost of qPCR so that is widely accessible in STH endemic countries.

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Section: Diagnostic Sensitivity and Methodology Testingmentioning

confidence: 99%

The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration

et al. 2020

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“…Previously, we described a pipeline for the identification of high-copy number repetitive DNA elements for use as semi-quantitative real-time PCR (qPCR) targets for the detection of various STH species [20–21]. These targets, identified utilizing next-generation sequencing (NGS)-based analysis tools, have facilitated improved sensitivity and specificity of detection, leading to their adoption in various diagnostic efforts and operational research (OR) studies such as the DeWorm3 cluster randomized trials [11, 14, 22]. Despite the availability of such tools, to date, the qPCR-based detection of A .…”

Section: Introductionmentioning

confidence: 99%

Targeting a highly repeated germline DNA sequence for improved real-time PCR-based detection of Ascaris infection in human stool

et al. 2019

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Background With the expansion of soil transmitted helminth (STH) intervention efforts and the corresponding decline in infection prevalence, there is an increased need for sensitive and specific STH diagnostic assays. Previously, through next generation sequencing (NGS)-based identification and targeting of non-coding, high copy-number repetitive DNA sequences, we described the development of a panel of improved quantitative real-time PCR (qPCR)-based assays for the detection of Necator americanus , Ancylostoma duodenale , Ancylostoma ceylanicum , Trichuris trichiura , and Strongyloides stercoralis . However, due to the phenomenon of chromosome diminution, a similar assay based on high copy-number repetitive DNA was not developed for the detection of Ascaris lumbricoides . Recently, the publication of a reference-level germline genome sequence for A . lumbricoides has facilitated our development of an improved assay for this human pathogen of vast global importance. Methodology/Principal findings Repurposing raw DNA sequence reads from a previously published Illumina-generated, NGS-based A . lumbricoides germline genome sequencing project, we performed a cluster-based repeat analysis utilizing RepeatExplorer2 software. This analysis identified the most prevalent repetitive DNA element of the A . lumbricoides germline genome (AGR, Ascaris germline repeat), which was then used to develop an improved qPCR assay. During experimental validation, this assay demonstrated a fold increase in sensitivity of ~3,100, as determined by relative Cq values, when compared with an assay utilizing a previously published, frequently employed, ribosomal internal transcribed spacer (ITS) DNA target. A comparative analysis of 2,784 field-collected samples was then performed, successfully verifying this improved sensitivity. Conclusions/Significance Through analysis of the germline genome sequence of A . lumbricoides , a vastly improved qPCR assay has been developed. This assay, utilizing a high copy-number repeat target found in eggs and embryos (the AGR repeat), will improve prevalence estimates that are fundamental to the programmatic decision-making process, while simultaneously strengthening mathematical models used to examine STH infection rates. Furthermore, through the identification of an optimal target for PCR, future assay development efforts will also benefit, as the identity of the optimized repeat DNA target is likely to remain unchanged despite continued improvement in PCR-based diagnostic technologies.

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“…Diagnostics based on targets discovered using this technique have proven useful in both past and ongoing field tests in Kenya (Pickering et al, 2019), Bangladesh (Benjamin-Chung et al, 2019), Ethiopia, Uganda, Timor Leste (Papaiakovou et al, 2017), Thailand (O’Connell et al, 2018), Liberia (Fischer et al, 2018), Japan, Benin, Malawi, India, and the Southern US, and have been adopted for use by large operational research efforts, such as the DeWorm3 cluster randomized trials (Ásbjörnsdóttir et al, 2018). However, testing biological samples, whether for diagnosing individuals or getting an overview of the epidemiological environment of a region, involves a myriad of factors, such as unexpected or emerging parasites, zoonotic infections, and misleading material in the samples.…”

Section: Resultsmentioning

confidence: 99%

“…In a similar case, the same ITS-based primer set (Llewellyn et al, 2016) was used to investigate Ancylostoma duodenale in a field study of a refugee population in Thailand (O’Connell et al, 2018). Since the most common human Ancylostoma parasite is A. duodenale , the results were initially believed to indicate A. duodenale infection.…”

Section: Resultsmentioning

confidence: 99%

“…These have traditionally been used for primer design and can give sensitive results depending on the size of the array (Verweij and Stensvold, 2014). However, the repeat is oftentimes conserved between species and even genera, and rDNA-based assays are often less specific than those designed from other repeat types (Pilotte et al, 2016; O’Connell et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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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

2019

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The balance of expense and ease of use vs. specificity and sensitivity in diagnostic assays for helminth disease is an important consideration, with expense and ease often winning out in endemic areas where funds and sophisticated equipment may be scarce. In this review, we argue that molecular diagnostics, specifically new assays that have been developed with the aid of next-generation sequence data and robust bioinformatic tools, more than make up for their expense with the benefit of a clear and precise assessment of the situation on the ground. Elimination efforts associated with the London Declaration and the World Health Organization (WHO) 2020 Roadmap have resulted in areas of low disease incidence and reduced infection burdens. An accurate assessment of infection levels is critical for determining where and when the programs can be successfully ended. Thus, more sensitive assays are needed in locations where elimination efforts are approaching a successful conclusion. Although microscopy or more general PCR targets have a role to play, they can mislead and cause study results to be confounded. Hyper-specific qPCR assays enable a more definitive assessment of the situation in the field, as well as of shifting dynamics and emerging diseases.

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Ancylostoma ceylanicum Hookworm in Myanmar Refugees, Thailand, 2012–2015

Abstract: This hookworm, uncommonly found in humans, has a higher cure rate than that for Necator americanus hookworm.

Cited by 16 publications

References 42 publications

The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration

The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration

Targeting a highly repeated germline DNA sequence for improved real-time PCR-based detection of Ascaris infection in human stool

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

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