The accuracy of nucleic acid amplification tests (NAATs) in detecting human intestinal nematode infections: A protocol for a systematic review and meta-analysis

Nalini Kaushalya Jayakody; Poornima Lakshini Kumbukgahadeniya; Anjana Silva; Nuwan Darshana Wickramasinghe; Susiji Wickramasinghe; Donald Peter McManus; Kosala Gayan Weerakoon

doi:10.1371/journal.pone.0278920

. 2022 Dec 12;17(12):e0278920. doi: 10.1371/journal.pone.0278920

The accuracy of nucleic acid amplification tests (NAATs) in detecting human intestinal nematode infections: A protocol for a systematic review and meta-analysis

Nalini Kaushalya Jayakody ^1,^2,^*, Poornima Lakshini Kumbukgahadeniya ², Anjana Silva ², Nuwan Darshana Wickramasinghe ³, Susiji Wickramasinghe ⁴, Donald Peter McManus ⁵, Kosala Gayan Weerakoon ^2,^5,^*

Editor: Hesham M Al-Mekhlafi⁶

PMCID: PMC9744273 PMID: 36508427

Abstract

Human intestinal nematode infections are a global public health issue as they can result in considerable morbidity in infected individuals, mainly in developing countries. These infections continue to go undiagnosed, as they tend to be mainly endemic in resource-poor communities where there is a shortage of experienced laboratory staff and relevant diagnostic technologies. This is further exacerbated by the nature of intermittent shedding of eggs and larvae by these parasites. Diagnostic methods range from simple morphological identification to more specialised high-throughput sequencing technologies. Microscopy-based methods, although simple, are labour-intensive and considerably less sensitive than molecular methods which are rapid and have high levels of accuracy. Molecular methods use nucleic acid amplification (NAA) to amplify the deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragments of the parasite to detect and determine its presence using different technologies (NAAT). They have increased the sensitivity of detection and quantitation of intestinal nematode infections, especially in low infection intensity settings. The absence of a gold standard test limits current diagnosis and, in turn, restricts intervention measures and effective control efforts. The objective of this review is to determine the accuracy of NAATs in detecting human intestinal nematode infections using Kato-Katz as the reference test for the most common soil-transmitted helminth (STH) infections and the scotch tape test for enterobiasis and Baermann method for strongyloidiasis. Relevant studies will be identified by searches in electronic databases. Two reviewers will independently screen the literature against eligibility criteria. The methodological quality of studies will then be appraised by two reviewers using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Discrepancies will be addressed by a third reviewer. The true positives, false positives, true negatives and false negatives of all the studies will be extracted into contingency tables. In paired forest plots, study-specific sensitivity and specificity with a 95 per cent confidence interval will be displayed. The systematic review of this protocol will report the diagnostic accuracy of currently available NAATs for the detection of human intestinal nematode infections. This will help healthcare providers and administrators determine the diagnostic method to be used in different clinical and preventive settings.

Trial registration: PROSPERO registration number for this protocol is CRD42022315730.

Introduction

Six intestinal nematodes that commonly infect humans are Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis and Enterobius vermicularis. Of these, A. lumbricoides, S. stercoralis, Ancylostoma spp., N. americanus, and T. trichiura are STHs [1, 2]. STH infections are the most common of the neglected tropical diseases (NTDs) globally. They cause an insidious impact on the deterioration of health and quality of life in impoverished populations, which leads to the perpetuation of poverty cycles [3]. Morbidity from STH infections becomes noticeable in a community only when a large number of people have moderate or high worm burdens, which usually happens when the prevalence is high. There has been an increased interest in controlling STH infections since the release of the world health organisation (WHO) road map to fight NTDs in 2012 [4]. The 2021 road map targets to reduce 75% of the Disability-Adjusted Life Years (DALYs) due to NTDs and to eradicate at least two NTDs from the world [5, 6].

Epidemiologically, STH infections are the most widespread among people residing in areas with insufficient sanitary facilities, low socioeconomic conditions and tropical climates [7, 8]. Even though frequent in low and middle-income countries, STH infections are also seen among vulnerable populations in high-income countries [9]. The presence of a wide range of symptoms and clinical signs, the challenges in nematode identification, and the scarcity of up-to-date epidemiological data, have made it difficult to arrive at precise estimates of the global burden due to intestinal nematodes. Insufficient disease monitoring systems have further exacerbated this issue. Consequently, estimating the disease burden depends on approximate values based on the best available data [10]. Current global figures show that around 1.5 billion people are diagnosed as suffering from STH infections, resulting in a disease burden of 5.2 million DALYs [11]. Over 267 million preschoolers and 568 million schoolchildren live in communities where these parasites are heavily transmitted and require treatment and preventive measures [12, 13]. S. stercoralis alone affects around 600 million individuals worldwide [14]. It is estimated that STH species infect around 361 million South Asians in 2019 [15]. Intestinal nematode infection burdens are higher in Asia due to the wet and hot climate, lack of access to clean drinking water, poor sanitation and sub-standard hygienic practices [16, 17]. In a national survey undertaken in 2019, the island-wide prevalence of STH infection was 0.97% (95% CI 0·63–1·48) among Sri Lankan schoolchildren aged 5–7 years. The prevalence of high-risk populations was greater than the national prevalence, which was 2.73% (0.75–6.87) in urban slum communities and 9.02% (4.29–18.0) in plantation sector communities [18]. However, these assessments are probably an underestimate of the true picture because they were calculated using microscopy-based methods, which have limited sensitivity in diagnosing intestinal nematode infections [19]. Even though microscopic methods have recognised low sensitivity, they are used in many instances as they are relatively simple and quick to perform, inexpensive and facilitate direct visualisation of nematode eggs and larval morphology. Individuals with high-intensity infections have higher morbidity, such as stunted physical growth and impaired cognition [17], whereas subjects with low-intensity infections usually do not have any symptoms. Data from the past ten years show a reduction in the STH infection prevalence in upper-middle-income countries, shifting the disease burden to lower-middle-income and low-income countries [20]. DALYs due to STH infections understate the accurate burden of disease, mainly to its incorrect attribution of hookworm-induced anaemia [1, 20].

Diagnostic methods for intestinal nematodes range from simple morphological identification to more specialised technologies, which include microscopic, serological and molecular-based diagnostics. Microscopic diagnostics include methods such as direct wet mount, Kato-Katz, formol ether concentration, spontaneous tube concentration, agar plate culture, water emergence method for detecting Strongyloides larvae in stools, Baermann technique, Harada-Mori technique, merthiolate iodine formaldehyde concentration, flotation procedures, McMaster’s method, FLOTAC and Mini-FLOTAC techniques, Stoll’s dilution egg counting technique.

NAATs are different molecular methods designed to amplify specific DNA or RNA fragments into a higher number of copies in order to determine and characterize their presence [21]. Types of NAATs used for the diagnosis of intestinal nematode infections are polymerase chain reaction (PCR), including conventional single, nested, real-time, multiplex PCR and digital PCR, isothermal assays like loop-mediated isothermal amplification (LAMP) quantitative nucleic acid sequence-based amplification (QT-NASBA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), strand invasion based amplification (SIBA), multiple displacement amplification (MDA), and more recently, cell-free DNA detection [22, 23]. They have substantially increased the sensitivity of detection and quantitation of intestinal nematode infections, especially in low infection intensity settings. They are also highly specific as they can help differentiate between closely related, morphologically identical, human and zoonotic species compared to microscopic techniques which could easily misidentify these parasites [23].

However, various diagnostic methods differ in terms of relevance, cost-effectiveness, ease of operation, sensitivity and specificity [13, 24, 25]. Microscopy has been the most extensively used tool for detecting, recognising, and quantifying parasites for many decades [26]. Currently, the WHO-recommended "gold standard" for the identification of STH eggs is the Kato-Katz method [27]. While parasite microscopy is specific, its sensitivity is dependent on the infection intensity and the time of specimen collection. As nematode eggs can be difficult to detect and identify in low-intensity infections [28], concentration methods are widely used in diagnosis. As nematode eggs have many morphological similarities, microscopy-based detection remains difficult. To be successfully controlled, NTDs must be targeted in an integrated approach [29]. Effective diagnosis should be precise, simple, inexpensive, and the procedures must provide results in a timely manner. Such methods are important in treatment and control programmes, during breakpoints in transmission, and in surveillance for reemergence when possible ’elimination’ targets have been achieved [26]. In recent years, there has been a growing effort in some high-income countries to use molecular techniques for STH infections in epidemiological studies and individual patient diagnosis [30, 31]. The main challenge in assessing the accuracy of molecular tests stems from the lack of an optimal gold standard. Even though stool microscopic techniques are commonly used as reference standards, they have relatively low sensitivity. The goal of this review is to generate new statistical evidence for healthcare professionals, researchers, and control strategy developers to use in diagnosing human intestinal nematode infections. Ultimately, this will facilitate improved screening, surveillance and the control and management of human intestinal nematode infections.

Objective

To determine the diagnostic accuracy of currently available NAATs for human intestinal nematode infections using Kato-Katz as the reference test for the most common STH infections and Scotch tape test for enterobiasis and Baermann method for strongyloidiasis, in different health care settings.

Methods

As described in this protocol, the methodological approach to evidence searching and synthesis will follow Joanna Briggs Institute (JBI) guidelines on systematic reviews of diagnostic test accuracy [32]. In reporting the findings of this review, standards of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) will be adhered to [33]. The content of this protocol follows the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) recommendations [34].

In this review, an initial literature search will be followed by the screening and selection of relevant studies against the preset eligibility criteria. Data extraction, methodological quality assessment, data analyses, evidence generation and report compilation will follow in chronological order.

Eligibility criteria

Inclusion criteria

Diagnosis of interest. Human intestinal nematode infections (ascariasis, trichuriasis, hookworm infection, strongyloidiasis and enterobiasis).

Population. We will include studies from the community, residential settings, primary, secondary and tertiary care settings. Studies reporting on mixed populations, different geographical regions, and different socioeconomic and social backgrounds will also be included. We will include all asymptomatic and symptomatic people without any restriction on the disease severity. There will be no limitations for gender, age group, ethnicity or country of origin.

The index tests. NAATs: Conventional single PCR, nested PCR, real-time PCR, multiplex PCR, and isothermal amplifications assays including LAMP, NASBA, SDA, RPA, SIBA and MDA.

The reference test. As no gold standard test is available, we will use WHO recommendations for the review, using the Kato-Katz as the reference test [35–37] for A. lumbricoides, T. trichiura and hookworm infections. We will use Graham’s Scotch tape test for E. vermicularis infection, and the Baermann technique for S. stercoralis infection as the reference test.

Outcomes. The specificity and sensitivity of the index tests are the major outcomes. The probability that the index test will be positive in an infected person is referred to as sensitivity [38]. The probability that the index test will be negative in a non-infected person is known as specificity [38].

Exclusion criteria

Case series, commentaries and expert opinions.

Studies that have not investigated the diagnostic accuracy of the index tests.

Studies that have been undertaken on animals.

Search strategy

An initial limited search of the PubMed online database on the diagnostic test accuracy of available microscopic and molecular methods of human intestinal nematode infections will be carried out. Following the initial search, a review of the keywords found in the titles and abstracts of the extracted feature papers, as well as the key phrases describing the articles, will be performed. Then we will conduct another search using all recognised keywords and index terms within all included databases, which are PubMed, Google Scholar, CINAHL, Scopus, Trip, Web of Science, and Cochrane Library. Finally, additional articles will be sought from the reference list of retrieved studies. The search terms for PubMed is (((((((((((((("ascaris"[All Fields]) OR ("roundworm"[All Fields])) OR ("necator"[All Fields])) OR ("ancylostoma"[All Fields])) OR ("hookworm"[All Fields])) OR ("strongyloides"[All Fields])) OR ("threadworm"[All Fields])) OR ("trichuris"[All Fields])) OR ("whipworm"[All Fields])) OR ("enterobius"[All Fields])) OR ("pinworm"[All Fields])) OR ("soil transmitted helminth"[All Fields])) OR ("geohelminth"[All Fields])) OR ("intestinal nematode"[All Fields])) AND (((((((((((((((((((diagnos*) OR ("diagnosis"[All Fields])) OR ("detect"[All Fields])) OR ("screen"[All Fields])) OR (investigat*)) OR ("investigation"[All Fields])) OR ("polymerase chain reaction"[All Fields])) OR ("pcr"[All Fields])) OR ("molecular"[All Fields])) OR ("nucleic acid amplification"[All Fields])) OR ("naat"[All Fields])) OR ("isothermal amplification"[All Fields])) OR ("loop mediated isothermal amplification"[All Fields])) OR ("lamp"[All Fields])) OR ("microscopy"[All Fields])) OR (microscop*)) OR ("kato katz"[All Fields])) OR ("baermann technique"[All Fields])) OR ("scotch tape"[All Fields])) Filters: in the last 10 years, Humans. This approach was developed from earlier pilot searches with the identification of keywords from relevant articles and in consultation with experienced researchers. We will be reviewing the studies undertaken within the last ten years with no language restrictions. Duplicate records will be removed initially with the Mendeley reference management software [39] and then with Rayyan software [40]. The whole text of the identified articles will be examined, with the eligibility criteria to generate the list of final articles.

Study selection

Findings from the electronic database search will be transferred to Rayyan software [40] for screening and shortlisting based on the inclusion criteria, where any duplicate studies will be removed. The authors will first look at the keywords included in the title and the abstract. Full-text sources of evidence that are relevant will be examined in greater detail based on the inclusion criteria. Two reviewers will work independently to select sources. If there are uncertainties, a third reviewer will be consulted. We will document the rationale for excluding any full-text source information and report it in the systematic review. If a full literature search reveals novel diagnostics that meet the eligibility criteria, these will also be included in the review. This procedure will be recorded, and the methodology will be illustrated in a PRISMA flowchart.

Assessment of methodological quality

Two reviewers will independently evaluate the risk of bias in each included article and report it according to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool [41]. Discrepancies that occur during the process will be resolved by the opinion of a third reviewer. QUADAS-2 includes the risk of bias assessments over four key areas: the patient selection, the index test, the reference standard, and assessment flow and timing.

We will employ the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to evaluate the quality of the body of evidence [42]. Accordingly, the body of evidence will be graded as high, moderate, low, or very low quality if data is available [43]. Two reviewers will separately evaluate the body of evidence for each gradable outcome in accordance with the study limitations, consistency of results, directness of evidence, precision, and study design in order to arrive at a grading. Finally, we will put together a summary of the evidence table using the GRADE development tool.

Data extraction

We will develop a data extraction form using the JBI data extraction instrument as guidance, with modifications relevant to the review. Two reviewers will retrieve information individually, using the customised data extraction form. To ensure consistency, the data extraction protocol will be tested on the first five to ten articles. During data extraction, the customised data extraction format will be amended and revised as needed. Supplementary data from included studies may be retrieved as decided by the review team during the process following the JBI methodology [39]. The revisions will be described in detail in the systematic review report.

Data to be extracted falls into the following domains:

Study identification details: Authors, the year of publication, the country and the funding source.

Study methodological details: Sample size, study design, clinical setting, number of drop-outs with reason, diagnostic methods assessed, parasite species studied, assessment of co-infection and any other relevant details like interventions carried out if any.

Population characteristics: Socio-demographic variables (ethnicity, sex, age, religion, marital status, education, employment, income, migration history, household details), history of deworming, and the intensity of infection.

Index test (NAATs) characteristics: The type of test, test design, target selection, the procedure of sample preparation and DNA extraction, output variables produced, quality control measures that were applied during the procedure, sample storage and transport method, the time duration between processing and analysis, and the cost of the test. Depending on the performance of the NAAT analysis, we will rank them accordingly and give different weights during the overall analysis. To give different weights, we will use the GRADE classification [42] as detailed in the methodological quality assessment.

The reference test: As there is no universally accepted “gold” standard, we will use the Kato- Katz as the reference test for the most common STHs, as this test is generally used in published literature and also recommended by the WHO as the most appropriate reference for these species. The way the Kato-Katz has been performed in each study, i.e. number of stool samples taken from a subject for the performance of the test, duration between each stool sample collection, number of smears examined from each stool sample, modifications that were done to the standard protocol, details of the modifications, and quality control measures that were applied during the procedure will be extracted from the selected primary studies. Based on the performance of the Kato-Katz smear examination of the primary study, we will rank each study and give them different weights in the overall analysis. As the reference tests, we will employ the Baermann method for strongyloidiasis and Graham’s Scotch tape test for enterobiasis. The number of smears that were evaluated and the timeframe that the sample was collected will be extracted for the Scotch tape sampling. In the Baermann technique, details of the procedure and quality control measures applied will be extracted.

Outcome measures: From contingency tables, we will calculate true positives, false positives, true negatives and false negatives leading to sensitivity, specificity, negative predictive value, positive predictive value, positive likelihood ratio and negative likelihood ratio of index tests.

Statistical analyses and evidence synthesis

Each index test will be compared against a reference test if data are available. For each test, a true positive, true negative, false positive and false negative will be retrieved. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of each study will be presented with 95% confidence intervals. They will be shown in paired forest plots. From primary studies, we will estimate summary sensitivity and specificity using the bivariate model [32]. The bivariate model will directly analyse the association between sensitivity and specificity and consider the random effects to be due to study heterogeneity. According to the availability of data, we will compare index tests using the above-mentioned approach [32]. When heterogeneity is present, it will be visually evaluated by comparing the study results on the paired forest plot. Meta-regression analysis will be used to explore the heterogeneity if five or more studies are available. Review manager software (RevMan web and RevMan 5) [44] will be used to perform this meta-analysis.

Discussion

Human intestinal nematode infections give rise to a significant global disease burden and high morbidity. Specific and sensitive diagnostic methods are critical in accurately determining worldwide and community burdens and precisely evaluating infection intensities in order to direct effective treatment and other control measures and to track the progress of ongoing STH control programs. Unfortunately, STH infections continue to be underdiagnosed, as they generally tend to be endemic in resource-poor, marginalised communities where there is a lack of trained laboratory staff and limited access to relevant diagnostic procedures. This is further exacerbated by the intermittent shedding of eggs and larvae by STHs which can impact the sensitivity of tests thereby preventing accurate diagnosis. Despite considerable progress in developing effective diagnostic methods, we are still a long way from identifying a gold standard test. Copromicroscopy has been the most extensively used tool for parasite detection and quantification for decades. However, there is a pressing need to avoid repetitive faecal sampling and to employ diagnostic methods with increased levels of sensitivity to assist in the monitoring of control programs, particularly in regions where elimination is the goal. Highly sensitive PCR-based methods can aid in the accurate measurement of STH burdens as control programs are advanced and are extremely useful in detecting worm infections, both in low-endemic areas and in travellers returning from endemic areas. These molecular methods can also diagnose the emergence of new STH variants as they arise. The approaches and methods that will be utilised in guiding this systematic review are outlined in this protocol. This is an important phase in the review process because it ensures that the final review has been well-planned and recorded to ensure accountability and transparency. It will also allow future researchers to replicate the review procedures [34]. The primary goal of this systematic review is to evaluate NAATs available for detecting human intestinal nematodes, including their sensitivity and specificity, and their strengths and limitations. This review will be focused on evidence-based conclusions and will provide key up-to-date information for researchers, clinicians and policymakers on the performance of microscopic and molecular-based approaches for the diagnosis of human intestinal nematode infections.

Supporting information

S1 Checklist. PRISMA–P 2015 checklist.

Preferred reporting items for systematic review and meta-analysis protocols 2015 checklist: Recommended items to address in a systematic review protocol.

(PDF)

Click here for additional data file.^{(217.4KB, pdf)}

Data Availability

This protocol was created in April 2022. The review team will then begin implementing the protocol outlined in this document. No datasets were generated or analysed during the current study. Upon completion of the systematic review, all relevant data from this review protocol will be made public.

Funding Statement

This review is a part of an intestinal parasitoses community evaluation project funded by the National Research Council of Sri Lanka (NRC 20-118). However, this particular publication (review protocol) is not funded through this grant. The funder had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0278920.r001

Decision Letter 0

Hesham M Al-Mekhlafi

12 Aug 2022

PONE-D-22-16144

The accuracy of PCR-based methods in predicting human intestinal nematode infections: a protocol for a systematic review and meta-analysis of the accuracy of available diagnostic tests

PLOS ONE

Dear Dr. Weerakoon,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please find the comments provided by the reviewers below this email.

==============================

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Hesham

Hesham M. Al-Mekhlafi, PhD

Academic Editor

PLOS ONE

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"This review is a part of an intestinal parasitoses community evaluation project funded by the National Research Council of Sri Lanka (NRC 20-118). However, this particular publication (review protocol) is not funded through this grant. The funder had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

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Reviewers' comments:

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Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

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(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes a protocol to perform a systematic review, analysing the diagnostic accuracy of PCR-based methods for the detection of human intestinal nematodes. Taking into account the increasing use of nucleic acid amplification tests (NAAT’s) in the diagnosis of intestinal helminths, I think the authors have chosen a timely topic for their review. However, I see several flaws and inconsistencies in their approach.

First of all, what I understand from the methods section is that the Kato-Katz smear will be used as the reference test. Therefore, clarity should be given about the way the Kato-Katz has been performed in each study, i.e. how many smears have been examined, based on how many stool samples and were there appropriate quality control measures in place. Based on the described performance of the Kato-Katz smear examination the authors could rank each individual study included and give them a different weight in the overall analysis.

Furthermore, for Strongyloides and Enterobius, it is not clear to me how the authors want to compare the PCR outcome with Kato-Katz data. For these two species other microscopy procedures (Baermann, tape-test) are more appropriate.

In addition the authors should be more specific about the NAAT’s included. Even if limiting to a real-time PCR format, each test is different, be it either in design and target selection, as well as in procedures of sample preparation and DNA extraction, the output variable producing, and most importantly, the level of appropriate quality control measures implemented. How to interpret these differences between publications is not clarified in this protocol. The description of Line 187-188 I find therefore far from complete.

This lack of uniformity is not only a major struggle for the analysis of PCR performance, but also for serology. This brings me to another point. In the abstract I read: “We plan to analyse the efficacy of microscopic- and PCR-based approaches in detecting human intestinal nematode infections in different health care settings.” In the key-words I noticed “serology”, but nothing is mentioned in the abstract. Only from the search strategy (line 225) and description at line 181-186 I understand that several immune-diagnostic tests will be included as index test, including antibody tests and antigen tests. This sounds rather ambitious to me. The authors should be more clear what the relevance is of including immunodiagnostic test and how they want to summarise the different tests available. My advise is to leave the immunodiagnostic tests out of the analysis.

Also for the different microscopy-based procedures included, the descriptions are not very clear to me. In my opinion the protocol would improve if the focus would be on a comparison between real-time PCR as the index test and the most optimal microscopy procedure as a reference test. Concerning microscopy, this is Kato-Katz for most STH species and species-dedicated procedures for Strongyloides and Enterobius

Minor comments:

Abstract: the proposed reference test is not described (as it is in line 138-140).

Line 62: other Ancylostoma species should also be included .

Line 226: ELIZA should be ELISA.

Line 226: “immunofluorescent antibody” is not an appropriate search term.

Line 278: “The gold standard: As there is no universally accepted gold standard, we will use those in the published literature and the WHO recommended gold standard, which is Kato Katz.” I suggest to rephrase this as follows: “Reference test: As there is no universally accepted “gold” standard, we will use the Kato Katz as the reference test for the most common soil transmitted helminths, as this test is generally used in published literature and also recommended by the WHO as the most appropriate reference for these species.”

Reviewer #2: Title: The accuracy of PCR-based methods in predicting human intestinal nematode infections: a protocol for a systematic review and meta-analysis of the accuracy of available diagnostic tests.

Comments

This is a well thought out protocol, focusing on diagnostic techniques. It is well written with clear and concise methods which are adequate to realise the study goals. However, I wish to raise a few issues that the authors should consider before publication of the protocol.

1. From the study objective, and the narrative in the whole protocol, it is clear that the goal of the systematic review is to evaluate different diagnostic techniques/ approaches that are available for evaluation of human intestinal nematodes. However, the title of the study is more biased to PCR-based approaches which are not the primary focus of the study. I will suggest that the title is reviewed to capture the whole study and not part of the study.

2. In the objective (Abstract section), the authors states that the study will specifically focus on microscopic and PCR based approaches, however in the Eligibility criteria (index tests Page 7 and 8), they propose to include serology based assays. This is a key area in diagnosis of human nematodes and needs to come out well in the objectives and also in the title.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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Attachment

Submitted filename: PLOS ONE. Reviewers comments.docx

Click here for additional data file.^{(13.3KB, docx)}

PLoS One. 2022 Dec 12;17(12):e0278920. doi: 10.1371/journal.pone.0278920.r002

Author response to Decision Letter 0

23 Sep 2022

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf

and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authorsaffiliations.pdf

Our response – Revised as requested adhering to the style guidelines.

2. Thank you for stating the following financial disclosure:

“This review is a part of an intestinal parasitoses community evaluation project funded by the National Research Council of Sri Lanka (NRC 20-118). However, this particular publication (review protocol) is not funded through this grant. The funder had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Our response - We have included the relevant details of the financial disclosure in the cover letter.

Reviewers' comments:

Our response to reviewer # 1

We thank the reviewer for his kind comments and useful insights. We found them very helpful to improve our protocol for the systematic review.

1. First of all, what I understand from the methods section is that the Kato-Katz smear will be used as the reference test. Therefore, clarity should be given about the way the Kato-Katz has been performed in each study, i.e. how many smears have been examined, based on how many stool samples and were there appropriate quality control measures in place. Based on the described performance of the Kato-Katz smear examination the authors could rank each individual study included and give them a different weight in the overall analysis.

Our response - As suggested, we have updated the data extraction phase of the Reference test in the methods section, to include information on the performance of the Kato-Katz, i.e.

number of stool samples taken from a subject for the performance of the test, the duration between each stool sample collection, number of smears examined from each stool sample, whether any modifications were done to the standard protocol, and the important details of the modifications done, quality control measures applied during the Kato-Katz procedure. Accordingly, when we are carrying out the data extraction of the selected studies, we will extract all of the above-mentioned details from the primary study. Based on the performance of the Kato-Katz smear examination of the primary study, we will rank each study and give them different weights in the overall analysis. (Line 241-247)

2. Furthermore, for Strongyloides and Enterobius, it is not clear to me how the authors want to compare the PCR outcome with Kato-Katz data. For these two species other microscopy procedures (Baermann, tape-test) are more appropriate.

Our response - We have added the Baermann test and the Graham Scotch tape tests as reference tests for strongyloidiasis and enterobiasis, respectively. (Line 159,160,247-252)

3. In addition the authors should be more specific about the NAAT’s included. Even if limiting to a real-time PCR format, each test is different, be it either in design and target selection, as well as in procedures of sample preparation and DNA extraction, the output variable producing, and most importantly, the level of appropriate quality control measures implemented. How to interpret these differences between publications is not clarified in this protocol. The description of Line 187-188 I find therefore far from complete. This lack of uniformity is not only a major struggle for the analysis of PCR performance, but also for serology.

Our response - As proposed, we have revised the Index test section of the data extraction to include the information on the NAATs, i.e test design, target selection, the procedure of sample preparation and DNA extraction, output variables produced, whether appropriate quality control measures were applied during the procedure Depending on the performance of the NAATs analysis, we will rank them accordingly and give different weights during the overall analysis. ( Line 232-237)

4. This brings me to another point. In the abstract I read: “We plan to analyse the efficacy of microscopic- and PCR-based approaches in detecting human intestinal nematode infections in different health care settings.” In the key-words I noticed “serology”, but nothing is mentioned in the abstract. Only from the search strategy (line 225) and description at line 181-186 I understand that several immune-diagnostic tests will be included as index test, including antibody tests and antigen tests. This sounds rather ambitious to me. The authors should be more clear what the relevance is of including immunodiagnostic test and how they want to summarise the different tests available. My advise is to leave the immunodiagnostic tests out of the analysis.

Our response - As suggested, we have dropped the analysis of immunodiagnostic tests from the protocol. As a result, the exclusion of the serology from the abstract and objectives is now addressed. Accordingly, we have removed “serology” from the keyword list and the search string.

5. Also for the different microscopy-based procedures included, the descriptions are not very clear to me. In my opinion the protocol would improve if the focus would be on a comparison between real-time PCR as the index test and the most optimal microscopy procedure as a reference test. Concerning microscopy, this is Kato-Katz for most STH species and species-dedicated procedures for Strongyloides and Enterobius

Our response - As suggested, we have removed all the other microscopic diagnostic methods as index tests.

6. Abstract: the proposed reference test is not described (as it is in line 138-140).

Our response - The reference tests were described in the abstract as suggested. (line 34-37)

7. Line 62: other Ancylostoma species should also be included.

Our response – Revised as suggested (revised line 54)

8. Line 226: ELIZA should be ELISA.

Our response - Serology was removed from the protocol. Accordingly, ELISA was also removed from this line.

9. Line 226: “immunofluorescent antibody” is not an appropriate search term.

Our response - As serology was removed from the protocol, the immunofluorescent antibody was also removed from this line.

10. Line 278: “The gold standard: As there is no universally accepted gold standard, we will use those in the published literature and the WHO recommended gold standard, which is Kato- Katz.” I suggest to rephrase this as follows: “Reference test: As there is no universally accepted “gold” standard, we will use the Kato -Katz as the reference test for the most common soil transmitted helminths, as this test is generally used in published literature and also recommended by the WHO as the most appropriate reference for these species.”

Our response - This line was revised as suggested. ( revised line 238- 241)

Comments

Our response to reviewer # 2

We thank the reviewer for appreciating our work and value the comments and suggestions given by the reviewer. Our responses to the comments are stated below.

Our response - As the first reviewer has suggested, we have taken serology out of our protocol as an index test. So we did not include any serology-related details in the abstract. Therefore, we feel that there is no necessity of amending the title.

Our response - As stated above, we have taken serology out of our protocol as an index test. Thus, we did not include any serology-related information in the abstract and the objectives.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(22.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0278920.r003

Decision Letter 1

Hesham M Al-Mekhlafi

12 Oct 2022

PONE-D-22-16144R1The accuracy of nucleic acid amplification tests (NAATs) in detecting human intestinal nematode infections: a protocol for a systematic review and meta-analysisPLOS ONE

Dear Dr. Weerakoon,

==============================Please find few minor comments by the reviewers at the end of this email. Moreover, please prepare the list of references according to the journal's instruction and style.==============================

Please submit your revised manuscript by Nov 26 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Hesham

Hesham M. Al-Mekhlafi, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

Reviewer #1: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I read the rebuttal and reviewed the changed made in the manuscript. In my opinion the authors responded well to all points mentioned by the reviewers. In particular I appreciate the more focused approach of the protocol.

Still, I have a number of technical comments. Most of these comments relate to the NAAT-procedures. The authors could be more precise in their wording and definitions at several points throughout the manuscript.

The line numbers mentioned refer to the manuscript including track changes.

Abstract (line 40): “… to more specialised high sequencing technologies such as the NAAT.” It is not clear to me what the authors try to say here. NAAT is a very broad definition of all tests related to nucleic acid amplification. Consequently “the” (?) NAAT is not an example of sequencing. More the other way around. Furthermore, the abbreviation NAAT is explained in the title. Still this should be repeated in the abstract (and in the introduction, see below).

Abstract (line 53): indeed, the details of the data bases are not so relevant for an abstract.

Objectives (line 151-152): I agree that the objectives improved by using NAATs and not “PCR-based methods”. But at the introduction section the terminology of NAATs have not been well introduced. In line 121-122 PCR is introduced as a method, but nothing is mentioned about other NAAT-methods, including isothermal amplification procedures, such as LAMP.

Inclusion criteria (line 187-188): LAMP is one example of several isothermal amplification procedures. It is not clear to me why only LAMP is mentioned an no other methods.

Search strategy (line 237-238): again it is not clear to me why only “loop mediated isothermal amplification” OR “LAMP” is included in the search terms for PubMed. I suggest to include the term “isothermal amplification” as well.

Data extraction (line 326-327): I appreciate the inclusion of a weight-factor, based on the quality of the used diagnostic procedures. But the description how this is done is completely lacking.

Data extraction (line 346-347): ”…the number of smears taken”. Please explain. I have never heard of “smears” in the context of a Baermann procedure. In addition ‘the standard technique” is not existing for the Baermann. Rather different approaches have been described.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

PLoS One. 2022 Dec 12;17(12):e0278920. doi: 10.1371/journal.pone.0278920.r004

Author response to Decision Letter 1

22 Nov 2022

Response to reviewers: Title: The accuracy of nucleic acid amplification tests (NAATs) in detecting human intestinal nematode infections: a protocol for a systematic review and meta-analysis

To,

The Editor/Reviewers,

PLOS ONE

Date: November 22, 2022

Subject: Addressing Editor’s/Reviewers’ Comments for PONE-D-22-16144

We appreciate the editor's and the reviewers' constructive suggestions on our revised protocol. We have attempted to address all the issues brought up by the academic editor and the reviewers. We believe that all the issues are satisfactorily addressed.

Sincerely on behalf of all the authors

Prof Kosala Weerakoon

Journal Requirements:

1. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response – The modifications we made to the reference section are listed below.

We have added new references numbering 21, 22, 23, 42 and 43 and accordingly references 39 and 41 of the previous list were removed to avoid repetition. We have not added any retraced papers.

Our response to reviewer # 1

We appreciate the reviewer's thoughtful remarks and insightful observations. They were incredibly useful to us in enhancing our systematic review protocol.

The line numbers we've provided correspond to those in the clean manuscript.

Reviewer #1: I read the rebuttal and reviewed the changes made in the manuscript. In my opinion the authors responded well to all points mentioned by the reviewers. In particular I appreciate the more focused approach of the protocol.

The line numbers mentioned refer to the manuscript including track changes.

1. Abstract (line 40): “… to more specialised high sequencing technologies such as the NAAT.” It is not clear to me what the authors try to say here. NAAT is a very broad definition of all tests related to nucleic acid amplification. Consequently “the” (?) NAAT is not an example of sequencing. More the other way around. Furthermore, the abbreviation NAAT is explained in the title. Still this should be repeated in the abstract (and in the introduction, see below).

Response – We have explained the NAATs in the abstract and also in the introduction section (lines 32-36, 109-121)

Abstract (lines32-36)

“Molecular methods use NAA to amplify the deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragments of the parasite to detect and determine its presence using different technologies (NAAT). They have increased the sensitivity of detection and quantitation of intestinal nematode infections, especially in low infection intensity settings.”

Introduction (lines 109 -121)

“NAATs are different molecular methods designed to amplify specific DNA or RNA fragments into a higher number of copies in order to determine and characterize their presence. Types of NAATs used for the diagnosis of intestinal nematode infections are polymerase chain reaction (PCR), including conventional single, nested, real-time, multiplex PCR and digital PCR, isothermal assays like loop-mediated isothermal amplification (LAMP) quantitative nucleic acid sequence-based amplification (QT-NASBA), strand displacement amplification (SDA), Recombinase polymerase amplification (RPA), Strand invasion based amplification (SIBA), Multiple displacement amplification (MDA), and more recently, cell-free DNA detection. They have substantially increased the sensitivity of detection and quantitation of intestinal nematode infections, especially in low infection intensity settings. They are also highly specific as they can help differentiate between closely related, morphologically identical, human and zoonotic species compared to microscopic techniques which could easily misidentify these parasites”

2. Abstract (line 53): indeed, the details of the data bases are not so relevant for an abstract.

Response – As suggested, we have removed the details of the databases from the abstract

3. Objectives (lines 151-152): I agree that the objectives improved by using NAATs and not “PCR-based methods”. But at the introduction section the terminology of NAATs have not been well introduced. In line 121-122 PCR is introduced as a method, but nothing is mentioned about other NAAT-methods, including isothermal amplification procedures, such as LAMP.

Response – We have introduced the word NAAT and provided details of the NAATs carried out in the diagnosis of human intestinal nematode infections. (lines 109-121)

Introduction (lines 109 -121)

4. Inclusion criteria (lines 187-188): LAMP is one example of several isothermal amplification procedures. It is not clear to me why only LAMP is mentioned and no other methods.

Response – As proposed, we have included other isothermal amplification methods to the inclusion criteria other than LAMP (lines172 and 173)

Inclusion criteria (lines 172 and 173)

“NAATs: Conventional single PCR, nested PCR, real-time PCR, multiplex PCR, and isothermal amplifications assays including LAMP, NASBA, SDA, RPA, SIBA and MDA”

5. Search strategy (lines 237-238): again it is not clear to me why only “loop-mediated isothermal amplification” OR “LAMP” is included in the search terms for PubMed. I suggest to include the term “isothermal amplification” as well.

Response – As suggested, we have included “isothermal amplification” to the search string (lines 206)

6. Data extraction (lines 326-327): I appreciate the inclusion of a weight-factor, based on the quality of the used diagnostic procedures. But the description of how this is done is completely lacking.

Response – We will use GRADE classification to give different weights to the studies based on the quality of the diagnostic procedure. In the methodological quality assessment section, we have introduced the term GRADE and given the details of the classification and how it will be utilized to give different weights to the studies based on the quality of the diagnostic procedure employed. (lines 233-239)

(Lines 233-239)

“We will employ the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach to evaluate the quality of the body of evidence. Accordingly, the body of evidence will be graded as high, moderate, low, or very low quality if data is available. Two reviewers will separately evaluate the body of evidence for each gradable outcome in accordance with the study limitations, consistency of results, directness of evidence, precision, and study design in order to arrive at a grading. Finally, we will put together a summary of the evidence table using the GRADE development tool.

6. Data extraction (line 346-347): ”…the number of smears taken”. Please explain. I have never heard of “smears” in the context of a Baermann procedure. In addition ‘the standard technique” is not existing for the Baermann. Rather different approaches have been described.

Response – We have removed “the number of smears taken” and “the standard technique” in the data extraction section of the Baermann test and included the “details of the different approaches of the Baermann test”.(lines 277 and 278)

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PLoS One. doi: 10.1371/journal.pone.0278920.r005

Decision Letter 2

Hesham M Al-Mekhlafi

24 Nov 2022

The accuracy of nucleic acid amplification tests (NAATs) in detecting human intestinal nematode infections: a protocol for a systematic review and meta-analysis

PONE-D-22-16144R2

Dear Dr. Weerakoon,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Hesham

Hesham M. Al-Mekhlafi, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

This version has addressed all comments highlighted by the reviewers.

I would like to take this opportunity to express my deepest sympathy and condolences on the loss of Prof. Donald McManus of QIMR Berghofer Research Institute, Australia, to his family, friends, students, colleagues and the science community. Prof. Donald McManus was an esteemed parasitologist who dedicated his life to the prevention and elimination of neglected tropical diseases, and has made incredible contributions to the field of parasitology. Rest in paece!

Hesham Al-Mekhlafi

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0278920.r006

Acceptance letter

Hesham M Al-Mekhlafi

2 Dec 2022

PONE-D-22-16144R2

The accuracy of nucleic acid amplification tests (NAATs) in detecting human intestinal nematode infections: a protocol for a systematic review and meta-analysis

Dear Dr. Weerakoon:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

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S1 Checklist. PRISMA–P 2015 checklist.

Preferred reporting items for systematic review and meta-analysis protocols 2015 checklist: Recommended items to address in a systematic review protocol.

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Data Availability Statement

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PERMALINK

The accuracy of nucleic acid amplification tests (NAATs) in detecting human intestinal nematode infections: A protocol for a systematic review and meta-analysis

Nalini Kaushalya Jayakody

Poornima Lakshini Kumbukgahadeniya

Anjana Silva

Nuwan Darshana Wickramasinghe

Susiji Wickramasinghe

Donald Peter McManus

Kosala Gayan Weerakoon

Roles

Abstract

Introduction

Objective

Methods

Eligibility criteria

Inclusion criteria

Exclusion criteria

Search strategy

Study selection

Assessment of methodological quality

Data extraction

Statistical analyses and evidence synthesis

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Hesham M Al-Mekhlafi

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Author response to Decision Letter 0

Decision Letter 1

Hesham M Al-Mekhlafi

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Author response to Decision Letter 1

Decision Letter 2

Hesham M Al-Mekhlafi

Roles

Acceptance letter

Hesham M Al-Mekhlafi

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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