This pilot study aimed to:

1. assess the feasibility of designing and delivering a technically and logistically challenging EQAS for the detection of Schistosoma and STHs, including Strongyloides in both preserved stool and DNA samples
2. evaluate and compare the diagnostic performance of laboratories, and
3. to gain insights in the diversity of the different NAAT protocols used

In order to do so, a panel of 12 validated stool samples and 8 DNA samples were distributed among participating laboratories across the globe, after which laboratories performed their respective in-house DNA extraction and NAAT protocols. Later, they reported the test results and completed a questionnair on their methodology. Finally, all results were analyzed and feedback on diagnostic performance was provided. 

The results revealed that although most laboratories performed well, there were clearly some laboratories that may need to improve their test protocol. It was also noteworthy that nearly all laboratores were applying a different protocol for their respective NAAT, highlighting a highly fragmented diagnostic landscape in need of some standardization. Further research should aim to verify whether this has an impact on the performance of the diagnostic outcomes.

From today onwards, SKML plans to provide a HEMQAS open to laboratories from all over the world. (more info)

You can now read the uncorrected Proof of the manuscript HERE!

The main objective of the project is to pilot a surveillance system to assess anthelmintic drug efficacy and the emergence of anthelmintic resistance in eight countries were preventive chemotherapy (PC) coverage has been high for at least five years.

It is expected that the results will provide important information on (i) the current STH prevalence and intensity of infections after multiple rounds of intense PC; (ii) global patters of efficacy of benzimidazole drug administered in the control program and (iii) the presence of genetic markers associated with benzimidazole drug resistance. Besides the research findings, this study will also form an important resource for countries that want to establish their own surveillance system and for future research on the molecular detection of AR.

This detailed description of the study rationale, study methods and plan of analysis presented in this manuscript are thought to form a valuable reference document for when study results are published in the future. What’s more, we also included a substantial amount of additional information, comprising detailed Standard Operating Procedures, summarized study protocols and informed consent forms in different languages (English, French and Spanish) to expand the scope of possible users. We therefore believe that the current protocol is of important interest for the whole community involved in monitoring the global STH status.

Happy reading from the Starworms team!

PDF of Protocol Paper

• Provide an overview of the different investments of the Bill & Melinda Gates Foundation to monitor / delay anthelmintic resistance
• Present and discuss the results on the diagnostic performance of molecular tools to assess single nucleotide polymorphisms in the beta-tubulin gene of human soil-transmitted helminths
• Present and discuss the current evidence of single nucleotide polymorphisms in the beta-tubulin gene in human soil-transmitted helminths
• Provide an update on the efficacy of drugs in countries where control programs reached high coverage for long time of period
• Work out an action plan for the analysis of the worms collected during the expulsion studies in Pemba Island (Tanzania) and Jimma Town (Ethiopia)

The Starworms team wishes to thank all participants of the meeting for the fruitful discussions and looks forward to a continued collaboration.

Given the many historic treatments with reported coverage of >90%, this district was of interest to evaluate the efficacy of the drugs used in the program. To do so, we planned a drug efficacy trial in the local schools of Sirajganj district.

The trial started on the 7th of September 2019. The field team (Pic 1) is evaluating the prevalence and intensity of STH infection in SAC in a dozen schools using duplicate Kato-Katz. Afterwards; all children receive a single dose of 500mg of Mebendazole as treatment (Pic 2). Fourteen to 21 days later; the children that were positive for any STH will be re-examined to be able to calculate the egg-reduction rate and evaluate the efficacy of the drugs.

We are looking forward to receive the findings of this study somewhere in the beginning of October. Right before the second deworming round of the year will take place in the thousands of little schools across this rapidly developing country.

The release of this collection coincides with the publication of two nice manuscripts describing the results of the work performed during Work Package 1 of the Starworms Project. The first manuscript focuses on the therapeutic efficacy of Albendazole as measured by single and duplicate Kato-Katz, Mini-FLOTAC, FECPAKG2 and qPCR. The second manuscript evaluates the diagnostic efficacy of these 5 different diagnostics.

Two earlier published manuscripts on the Starworms Work Package 1 protocol and the FECPAKG2 method are also included in the collection at this time.

We are looking forward to expand this collection very soon with a number of additional manuscript of importance to the STH community.

Happy reading!

The Starworms Team.

Bruno, Piet and Johnny

In the year 2017, globally, 72.7% of the 3452 implementation units reached an effective coverage of >75%, reaching a total global coverage of 69%. For Pre-School Aged Children, a global coverage of 69% was reached. For School Aged Children, global levels of coverage were 68.8%. The presented numbers show a significant improvement in coverage, in line with the NTD road map targets for 2020.

However, it is of note that  the current NTD road map does not target adults at risk for Schistosoma or women of childbearing age for STH. As a result, PC coverage rates for those groups are very low because the drugs are not donated. This is a challenge that should be addressed in the future.

Lets keep up the good work!

The official protocol on the first workpackage of the Starworms project is now published in PlosNTD.

In this protocol paper we delve deeper into the why and how of the starworms project and we discuss the approach and plan of analysis for the first work package of this project. It is an excellent work of reference since it includes many of our standard operating protocols and project record forms.

Download your copy by clicking on this link:

STARWORMS WP1 PROTOCOL

Upon deeper evaluation of the data collected and provided in this paper an important methodological issue came to light that could provide a more straightforward explanation as to why a reduction in effecacy and cure rates were observed. The authors namely reported that the post-treatment examination was performed between 7 and 10 days after the treatment with ALB. At that time, complete expulsion of adult worms and/or parasite eggs can however not be ensured and would likely lead to misleading conclusions regarding drug efficacy.

Although the time point at which post-treatment samples are collected is crucial to readily interpret the drug efficacy, empirical evidence for the optimal timing of post-treatment sampling remains scarce for all STHs. In this light, Levecke et al. followed with the publication of the results of an expulsion study performed by Easton and colleagues in 19 Kenyan individuals treated with ALB. (Find paper HERE) There it was apparant that it takes up to 10 days for all infected individuals to clear the adult worm load and that approximately 20% of all worms is being expelled between day 7 and 10 post treatment.

The barplot represents the number of adult Ascaris worms recovered from 21 Kenyan patients treated on day 0 with 400mg Albendazole over a time span of 10 days post treatment  (white: female; black:male and grey: unknown sex)

Based on these findings, it is recommended to wait at least 14 days after an ALB treatment before conducting follow-up egg counts.Any sampling performed before this time point may result in biased ERR estimates, due to release of residual eggs from moridund or degenerating worms.

PRESENTATIONS

1. How to ensure the efficacy of drugs in soil-transmitted helminth control programs? by Bruno Levecke (Ghent University)
2. Comparison of 4 diagnostic tools for the assessment of drug efficacy of a single dose albendazole against soil-transmitted helminths by Johnny Vlaminck (Ghent University)
3. Molecular diagnosis for benzimidazole resistance-associated SNPs in human soil-transmitted helminths By Roger Prichard (McGill University)

POSTERS:

1. The comparison of preservation and DNA extraction methods for the molecular detection and quantification of soil-transmitted helminth infection in stool (Ayana et al.)
2. Optimizing FECPAKG2 for the quantification of soil-transmitted helminth eggs in human stool (Ayana et al.)
3. First international external quality assessment of nucleic acid amplification tests for the detection of soil-transmitted helminths – the study protocol (Cools et al.)
4. ParaDesign 2.0: an improved E-tool to identify the most cost-effective survey design to monitor deworming programs targeting soil-transmitted helminthiasis (Levecke et al.)
5. ParaDrug: an E-tool to analyze and report drug efficacy data in preventive chemotherapy programs targeting schistosomiasis and soil-transmitted helminthiasis (Levecke et al.)

Annual or bi-annual mass drug administration (MDA) with ivermectin is the recommended strategy to control onchocerciasis. MDA is likely to exert selection pressure on these parasites, and phenotypic and genetic changes in several O. volvulus populations from Cameroon and Ghana—exposed to more than a decade of regular ivermectin treatment—have raised concerns that sub-optimal responses to treatment with ivermectin are becoming more frequent and may spread.

In the study by Doyle and coworkers, the authors the genetic diversity between 108 Onchocerca worms isolated from patients from Ghana and Cameroon was compared with different individual and/or community ivermectin treatment histories and different responses to treatment.

Genome-wide analyses revealed genetic variation that significantly differentiated worms isolated from good responders and suboptimal responders. These genomic differences were not randomly distributed throughout the genomes, but were clustered in 31 loci, with little overlap in position or gene content between countries. Genes previously suggested to be associated with suboptimal responses to ivermectin were largely absent in the loci.

The authors concluded that ivermectin responses are polygenically-determined quantitative traits, and that identical or related molecular pathways but not necessarily individual genes are likely to determine the ivermectin responses.

This new protocol describes both the Mini-FLOTAC apparatus as well as how to use of the Fill-FLOTAC should be used,  a closed system that  facilitates the performance of the first four consecutive steps of the Mini-FLOTAC technique: fecal sample collection and weighing, homogenization, filtration, and filling of the Mini-FLOTAC chambers. Using the prescribed protocol, a single sample prep is estimated to take approximately 12 minutes, which is a considerable improvement over the earlier developed FLOTAC.

During the Starworms trials, the number of STH eggs in human stool is also quantified using the Mini-FLOTAC. We have considerable experience with the technique and agree with the authors that this a highly sensitive coprological technique that can be easily performed in the setting of a field based lab after some basic training of the staff.

Here you can find the  Mini-Flotac protocol

At the summit, the Belgian federal minister for development co-operation Alexander De Croo shared the stage with Bill Gates and other CEO's. The minister announced that Belgium will invest a total of 25.3 million euros to wipe out sleeping sickness. The Bill and Melinda Gates Foundation will double that amount. "It must give a final push to eradicate the disease," said De Croo. The efforts will be managed by the Institute for Tropical Medicine (ITG) in Antwerp (Belgium) and will focus on the Democratic Republic of Congo, since more than 85 percent of the cases are reported in Congo. 

According to the World Health Organization (WHO), over 20,000 people worldwide are infected with sleeping sickness, which is transmitted by the tsetse fly. Over 50 million people are at risk of getting the disease. The fact that science has progressed in the fight against the disease in recent years has shown a sharp fall in the number of cases. By 2015, there were less than 3,000, or a 89 percent decline since 2000. It is the hope of De Croo and Gates that this new effort, led by the ITG, will deal a fatal blow to this devastating illness.

Picture of the large panel of stakeholders at the 2017 NTD summit in Geneva. Bill Gates sits on the far right. Federal minister of development co-operation Alexander De Croo is the 6th person from the right.

Top view of the first batch of samples selected for DNA extraction and qPCR analysis for STH.

The Kato-Katz technique for diagnosing helminth infections (mainly STH and Schistosoma mansoni) in the stool is well established and recommended by the World Health Organization. However, this technique has some issues: it is labor-intensive, requires expertise, needs to be performed quickly after stool collection and generally has a poor sensitivity to detect light infections. The cost of performing a single Kato-Katz is estimated between 2.67 and 12.48 US dollar. This cost is highly variable and depends on multiple factors, including but not limited to (i) collection method, (ii) number of samples and distance between collection sites, and costs for both (iii) personnel and (iv) the material.

It is put forward that investing in the development of new diagnostic techniques is necessary. This is especially relevant now, when changes in control strategies will become important as goals shift from morbidity control towards elimination of transmission. On the one hand, the development of more rapid and easy-to-use diagnostics is needed make it more accessible for non-trained health or education professionals. This would make it easier to integrate STH with other neglected tropical disease surveillance programs and therefore greatly reduce costs. On the other hand, more sensitive tests are needed to be able (i) to reduce sample size when prevalence has dropped after control efforts, (ii) to quickly detect resurgence where transmission was previously interrupted , and (iii) to quickly detect the development of possible anthelmintic resistance. Early detection of anthelmintic resistance will be crucial to allow for adjustments in control strategies.

It is important to stress that costs of diagnostic techniques will differ in different settings. It is essential to accurately capture the implementation costs of novel diagnostics and investigate what the key drivers of these costs are. In the Starworms project the costs of 3 different diagnostic techniques for the detection of eggs in the stool is assessed in 4 different endemic settings. Furthermore, these costs will be incorporated in ParaDesign 2.0. , a digital tool designed by our team to help with the selection of the best cost-effective trial design.

Overall, Turner et al. clearly stated that it is obvious that without new diagnostic methods, it will become very difficult for STH and Schistosoma control programmes to make the most cost-effective policy decisions, particularly as the goals shift towards elimination..

Read the Original paper here

Example of a LAMP assay. Tubes that light up under UV-radiotion are positive for a certain gene of interest.

Wild-type (WT) hookworm strains and strains with a mutation (MT) in the tubulin-gene, making these strains resitant towards benzimidazole.

-> Read more

Field team in Grade 2C of Dilfire school in Jimma, Ethiopia.

Lab technician from Jimma University analysing samples with the FECPAKG2 system.

The microscopists scanning Kato-Katz slides and Mini-FLOTAC devices for the presence of STH eggs.

All stool samples are stored in preservative for future parasite DNA extraction.

Currently the team is performing the double data entry of all raw data materials . Final datasets will be available soon for analysis.

1.  present the final protocol
2.  agree on the data management and data entry procedures
3.  outline the monitoring visits 
4.  demonstrate the diagnostic tools.

The morning of Day 1 was dedicated to provide an overview of the entire project, followed by a detail discussion on the study protocol of work package 1. In the afternoon the different study sites (Brazil, Ethiopia, Lao PDR and Tanzania) were presented and updates were shared on the Helminth Egg Automtic Counter (HEAD, software to automate egg counting) and the different diagnostic assays which are going to be applied in the project, including both the microscopic (FECPAKG2 and Mini-FLOTAC) and molecular based diagnostics (qPCR, pyrosequecning and LAMP).

Days 2 and 3 were dedicated to workshops on data management and data entry procedures, outline the monitoring visits and the diagnostic tools, including FECKPAKG2 and Mini-FLOTAC. As illustrated below, the particpants were invited to hands-on sessions.

Participants preparing and examining a stool sample with Mini-FLOTAC

These sessions revealed to most usefull, as they helped in 

• further simplifying the different forms
• indentifying potential obstacles in processing a large number of stool samples
• pinning down important gaps in standard operating

Participants preparing and examining a stool sample with FECPAKG2