In this study, we evaluated the implementation of diagnostic methods for S. stercoralis within the ongoing STH and schistosomiasis control programme in Rwanda, initiated in 2007. Participants’ compliance was excellent, with all individuals providing the required biological samples in sufficient volumes. All samples were processed using the planned techniques, from sample collection to testing, without any logistical or technical disruptions. The current study employed experienced technicians who are involved in STH and Schistosomiasis mapping studies as part of the NTD control programme in Rwanda. At the beginning of the study, technicians reported some difficulties implementing the Baermann and APC methods; these challenges were solved with practice.

Initially, the majority of technicians reported “insufficient previous training” as the main reason for difficulties encountered in the implementation of both methods. By the third evaluation timepoint, none of the steps were reported as “difficult” or “very difficult”, suggesting that a structured training programme (incorporating hands-on practice with real samples) significantly enhanced technicians’ competency over time. Other common reasons noted for technical difficulties included “technical problems” and “larvae identification”. Although cases of misidentification (i.e., the technician presenting a possible S. stercoralis larva, which was subsequently identified as something else by the supervisors) were not formally quantified in this study, supervisors reported an initial poor performance that improved gradually. Hence, we recommend that NTD national programmes should not underestimate the amount of time dedicated to tailored training for the correct application of S. stercoralis-specific coproparasitological techniques, particularly in areas where S. stercoralis is co-endemic with hookworm.

The prevalence of S. stercoralis reported in this study was comparable to the 1.9% observed in a previous study carried out in 2016 in Gisagara by faecal direct smear, which is a parasitological technique known for its exceedingly low sensitivity compared to Baermann and APC methods¹³. In the same study, a 17.4% prevalence was reported using horse blood agar culture, a method which is, however, not recommended for S. stercoralis detection⁷. Although in nine years the epidemiology of strongyloidiasis may have changed, the figures from 2016 and from this study differ too much to be attributable to individual treatment with ivermectin (no MDA with ivermectin has ever been done in Rwanda). Of note, ivermectin has never been used for clinical case management due to its unavailability in Rwanda until COVID-19 period, despite being recommended by the national treatment guidelines for strongyloidiasis, trichiuriasis and scabies cases. Furthermore, the 2016 study relied on visual detection of “larval tracks”, a characteristic exhibited also by other migrating larvae, suggesting potential misdiagnosis¹⁴.

As opposed to coproparasitological techniques, the use of the RDT was straightforward at all steps and with either blood or plasma samples. Most staff members were already familiar with similar tests such as those for malaria. An RDT would be particularly useful for fieldwork, and would not require long training for its deployment. No concerns were raised about waste disposal of the tests, contrary to a previous study where this aspect was flagged as an issue¹⁵. It is important to note that the RDT used in this study was a dipstick format, whereas the previous study used a plastic cassette.

The point-of-care format, the short time needed for training (one day or less), the possible use by staff with no specific laboratory expertise (i.e., possible use by community health workers), the short time to obtain the results (less than 1 h), the high throughput (at least ten tests per hour per tester) are among the characteristics of the RDT that comply with the recently issued target product profile (TPP) for S. stercoralis diagnostics¹⁶.

Overall, the prevalence data obtained by the RDT were in line with those from the faecal tests. However, the discrepancies between RDT and the faecal test results in SAC may be due to adults having more long-lasting infections, permitting enhanced antibody production compared to children. Further data are needed to explore these aspects, especially since SAC are commonly the target population for other STH prevalence surveys¹⁷. An evaluation of the RDT’s diagnostic accuracy, incorporating stool PCR as a reference standard, is currently underway.

Recently, a cost-effectiveness analysis was conducted to support the development of the TPP for diagnostics in S. stercoralis control programmes¹⁸. The analysis showed that an RDT could be more cost-efficient than Baermann, provided it demonstrated good specificity (at least 72% for adequate decision-making and 84% for ideal decision-making) and cost around one US dollar per test. The materials and equipment costs for the Baermann technique estimated in our study were comparable to those reported in Ecuador¹⁵, which informed the cost-effectiveness analysis. However, the RDT used in Ecuador was nearly twice as expensive as the one deployed in this study, highlighting variability in assay costs. Of note, neither RDT is available on the market yet, so their cost may change once commercially available. The APC was not included among the assays considered in that cost analysis; in this study, its cost was comparable to that of Baermann; both, in any case, costed less than two euros per test in terms of material and equipment needed per sample. When considering personnel costs in the particular setting of Rwanda for the processing of samples collected in one day of field work from reception of the sample to result (Supplementary File), the cost increase of integrating one S. stercoralis-specific assay to Kato Katz for STH and Schistosoma was less than 50% for RDT, while reached 65% and 70% for Baermann and APC, respectively. About one third of the total cost was due to equipment and materials, while two thirds were due to personnel, highlighting the importance of a thorough evaluation of total survey costs and cost-effectiveness analysis of the actual field logistic deployed, when planning an integrated control programme, even if using the same sample matrix.

This study has several limitations. Mass treatment with ivermectin was not recommended in either of the investigated Rwandan districts, as the prevalence of S. stercoralis infection was below the 10% threshold established for community-based surveys in the WHO guidelines^8,9. Consequently, one limitation of this study is our inability to provide information on potential challenges associated with implementing ivermectin distribution. The small number of S. stercoralis cases might also have impacted the training and its evaluation. Indeed, the low prevalence could have resulted in an increase of the time required for slide reading (more time is needed to read the whole sediment when no larvae are detected, compared to cases where larvae are found and the slide is therefore discarded after the first S. stercoralis larva identification), but also in a reduced attention from technicians, who might become gradually accustomed to examine and therefore expect mainly negative samples. Furthermore, technicians had only limited chance to examine S. stercoralis larvae from the study samples. A further limitation is that we did not quantify the proportion of larvae or larvae-like objects that were misidentified as S. stercoralis under microscopy, as this was technically challenging. Furthermore, we did not incorporate a feasibility assessment of PCR performance in a central laboratory or collect blood samples (e.g., dried blood spots) for laboratory-based seroassays, as the Rwanda NTD programme did not foresee the potential to implement these screening strategies. Moreover, a comprehensive cost assessment of an entire strongyloidiasis screening initiative integrated into a control programme for STH, or of extensive training for coproparasitological diagnosis of S. stercoralis, were beyond the scope of this study.

The strengths of this study include a formal assessment of the feasibility of each technique at multiple stages, allowing for a thorough evaluation of critical steps that may require retraining. Additionally, the participant sample size was sufficiently large to provide a representative estimate of S. stercoralis prevalence in each district, in accordance with the WHO recommendations.