E N'kaoua

S Attarian
email: shahram.attarian@ap-hm.fr

E Delmont

E Campana-Salort

A Verschueren

A.-M Grapperon

E Mestivier

M Roche

Immunoglobulin shortage: Practice modifications and clinical outcomes in a reference centre

Results. -Out of 142 patients, 111 (78%) had a modification of their IVIg treatment. We noted that 75 (68%) patients had a delay in IVIg treatment, 41 (37%) patients had a decrease in IVIg doses and 31 (28%) experienced IVIg treatment interruption. Thirty percent of patients for whom IVIg treatment was discontinued were switched to other treatments mainly plasma exchange (16%) or corticosteroids (13%). Switches to plasma exchange or corticosteroids were carried out in order to save immunoglobulins for patients who had no other alternatives. Fifty-eight (52%) patients presented a deterioration of their clinical score after IVIg treatment changes including 31 (28%) patients who had a moderate or a clinically significant deterioration. Concerning practice modifications, we noted a substantial though not significant decrease in median IVIg dose for myasthenia gravis and a significant increase in the delay between IVIg courses for chronic inflammatory demyelinating polyneuropathy and multifocal motor neuropathy (P = 0.011 and P = 0.018 respectively).

Conclusion. -Our study showed a rather important number of changes in IVIg treatment related to IVIg shortage during the period considered. These changes had a negative impact on the clinical status of some patients.

# 2021 Elsevier Masson SAS. All rights reserved.

1.

Introduction

Immunoglobulins (Ig) are therapeutic preparations obtained from blood plasma [START_REF] Guilpain | Effets immunomodulateurs des immunoglobulines intraveineuses[END_REF]. Ig use began in 1950, first by intramuscular injection, then at the beginning of the 80s intravenously in replacement therapy for primary immunodeficiency [START_REF] Mouthon | Indications et me ´canismes d'action des immunoglobulines intraveineuses dans les pathologies auto-immunes et inflammatoires syste ´miques[END_REF]. In 1981, Imbach et al. noticed their efficiency in treating immunological thrombocytopaenic purpura. This discovery made it possible to use Ig for autoimmune and inflammatory diseases [START_REF] Guilpain | Effets immunomodulateurs des immunoglobulines intraveineuses[END_REF][START_REF] Imbach | High-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood[END_REF].

For a few years now, the use of Ig has been steadily increasing worldwide. Over the last 15 years, the demand has risen three-fold [START_REF] Sewell | European consensus proposal for immunoglobulin therapies[END_REF][START_REF] Kerr | Is dosing of therapeutic immunoglobulins optimal?. A review of a three-decade long debate in europe[END_REF], with a great increase of consumption in emerging countries throughout Asia and the Middle East [START_REF] Gay-Crosier | Peut-on se passer des immunoglobulines polyclonales intraveineuses (IVIg)?[END_REF]. In France, the use of Ig increases also every year, with an average rise of about 10% per year [START_REF] Veran | La filie `re du sang en France[END_REF]. This phenomenon can be explained by expanded indications these last years, mainly in immunomodulatory therapy with over a hundred pathologies involved. Yet, few of these indications get a market authorisation; today off-label indications represent 50% to 70% of the demand for intravenous Ig (IVIg) [START_REF] Bayry | Shortage of human intravenous immunoglobulin-reasons and possible solutions[END_REF].

Recently, the third National Ig Database Report of the British National Health Service pointed out that neurologic indications are, at the moment, the main reason internationally for the use of IVIg [START_REF] Sewell | European consensus proposal for immunoglobulin therapies[END_REF].

Immunomodulatory doses of IVIg given for autoimmune or inflammatory diseases are four or five times higher than the doses used in replacement therapy for primary and secondary immune deficiency with a total dose of 1-2 g/kg of IgG injected within 2-5 days per month [START_REF] Elovaara | EFNS guidelines for the use of intravenous immunoglobulin in treatment of neurological diseases: EFNS task force on the use of intravenous immunoglobulin in treatment of neurological diseases[END_REF]. This is why the improvement and harmonisation of Ig use in neurology is a real concern within our university centre and requires an evaluation of professional practices.

This increasing demand associated with the limited supply of the raw material has caused problems of procurement and international shortage [START_REF] Sewell | European consensus proposal for immunoglobulin therapies[END_REF]. In France, the shortage was particularly worrysome at the beginning of 2018, related to production restrictions of the largest pharmaceutical group. These tensions were to be exacerbated in the coming months due to a shortage of blood and plasma collection because of the coronavirus disease 2019 (COVID-19) crisis.

A review on the impact of drug shortages on patient outcomes, published in 2019, reported that drug shortages had negative clinical, economic and humanistic consequences for patients. However, there were no reports on IVIg [START_REF] Phuong | The impacts of medication shortages on patient outcomes: a scoping review[END_REF].

The objective of our study was to assess the impact of IVIg shortage on patient care and to evaluate the clinical consequences.

2.

Patients and methods

Study design

This was a retrospective, single-centre study, conducted from October 2017 to October 2018, in the department of neuromuscular diseases at the University Hospital, la Timone, Marseille, France. We assessed patient data one year before the shortage, in 2016, and one year after the shortage, in 2019.

The study included all patients given IVIg for chronic inflammatory demyelinating polyneuropathy (CIDP), Lewis and Sumner syndrome (LSS), multifocal motor neuropathy (MMN), or myasthenia gravis.

We excluded: patients with incomplete data; deceased patients; patients not monitored during the period under consideration for indirect impact analysis.

Data were collected by AXIGATE1 (university centre database) and were supplemented with medical records and diaries in Pharma1 software (Computer Engineering, version 5.9). This software has been used in our hospital pharmacy since 2010 to store and record drugs delivered to inpatients and outpatients and in care units to track prescriptions and drug administration.

Data collection and analysis

IVIg treatment (IVIg type, dose and infusion regimen) and patient data (sex, weight, and disease) were anonymised to maintain confidentiality and collected in an Excel table.

When evaluating the impact of IVIg shortage, we noted three types of treatment changes: decrease in IVIg dose; discontinuation of IVIg treatment and; IVIg treatment delay.

IVIg treatment delay was defined as at least a 7-day delay between two courses for regimens scheduled for four weeks and at least a 3-day delay for regimens scheduled for less than four weeks. The events studied were the number of types of changes (decrease in IVIg dose, delay in IVIg treatment, discontinuation of IVIg treatment) for each patient to assess the impact on patients' IVIg courses.

The median IVIg dose and the median delay between two IVIg treatments were compared between 2016 and 2019, i.e. one year before shortage and one year after shortage, for each dysimmune neuromuscular disease separately, in patients followed between 2016 and 2019.

In addition, scores on the following clinical scales were collected for all patients: Rasch-built overall disability scale (RODS) and overall neuropathy limitation scale (ONLS) for CIDP and LSS, Rasch-built overall-disability scale (MMN-RODS) for MMN and myasthenia muscle scale for myasthenia gravis [START_REF] Vanhoutte | Rasch-built Overall Disability Scale for Multifocal motor neuropathy (MMN-RODS(#))[END_REF][START_REF] Van Nes | Rasch-built Overall Disability Scale (R-ODS) for immune-mediated peripheral neuropathies[END_REF]. Each patient's functional scores were averaged; then, deviation from the mean was determined after each prescription change. Clinical outcomes were evaluated for each patient by expert analysis (S.A).

The RODS was scored on 48 points and then recalculated on 100 points. The MMN-RODS was scored on 50 points and then recalculated on 100 points [START_REF] Vanhoutte | Rasch-built Overall Disability Scale for Multifocal motor neuropathy (MMN-RODS(#))[END_REF][START_REF] Van Nes | Rasch-built Overall Disability Scale (R-ODS) for immune-mediated peripheral neuropathies[END_REF].

For data analysis purposes, the patients were divided into three groups: Group 0: Low score deterioration, according to our considerations and those of the medical team, i.e. slight but stable discomfort: ONLS score (unchanged); RODS score (decrease < 4 points); myasthenia muscle score (decrease < 5 points); Group 1: Moderate deterioration: ONLS score (increase = 1 point); RODS score (decrease ! 4 and < 6 points); myasthenia muscle score (decrease ! 5 and < 10 points); Group 2: Clinically significant deterioration, according to our consideration: ONLS score (increase ! 2 points); RODS score (decrease ! 6 points); myasthenia muscle score (decrease ! 10 points).

For each patient with a score deterioration, a clinical validation was then made with the medical team neurologist to determine if the shortage caused the deterioration, thus defining patients affected by the IVIg shortage context.

We examined the time course of functional scores in all patients (with or without IVIg treatment changes) to evaluate the link between severe supply shortage and clinical outcomes in our hospital's centre for neuromuscular diseases, using statistical analysis to evaluate the association between IVIg treatment changes and clinical impact.

Statistical analysis

Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS) software (V.23) using the paired Student t-test, the Wilcoxon signed-rank test, or the x 2 test. A value of P < 0.05 was considered significant. Fig. 1 illustrates this process.

Results

Patient characteristics

During our analysis period, 142 patients were included (median weight 76 AE 25 kg, median age 61.5 AE 15.2 years).

Twenty patients were excluded from the first analysis due to incomplete data or death during the analysis period. Among the 142 patients, 47 (33%) had CIDP, 40 (28%) had LSS, 39 (28%) had MMN, and 16 (11%) had myasthenia gravis.

Table 1 lists patient characteristics.

3.2.

Impact on treatment Table 2 provides the distribution of IVIg treatment changes by dysimmune neuromuscular disease.

All IVIg treatment interruptions were maintained for at least one year. Nine (30%) of the patients for whom IVIg treatment was discontinued, were switched to other treatments: plasma exchange (PE) for five (16%) patients; corticosteroid therapy for four (13%) patients.

Among the patients whose IVIg therapy was delayed, the modified IVIg treatment schedule was maintained in 21 (28%). Among those whose IVIg dose was decreased, the modified dose was maintained in 14 (35%). Table 3 notes the maintenance of IVIg treatment changes.

Impact on medical practices

The IVIg supply shortage changed medical practices indirectly. For this part of the analysis, 51 patients were included and 104 were excluded.

There was no significant difference in median dose before (i.e. 2016) versus after (i.e. 2019) the shortage for CIDP and LSS, but there was a significant decrease in IVIg median dose for MMN: 1.83 (range 1.13-2.62) g/kg in 2016 versus 1.69 (range 1.13-2.03) g/kg in 2019 (P = 0.034). For myasthenia gravis patients, there was a non-significant decrease in median dose: 1.73 (range 1.64-1.9) g/kg in 2016 versus 1.15 (range 1-1.62) g/kg in 2019 (P = 0.423).

Median delay between IVIg courses was significantly increased for CIDP and MMN patients. For CIDP patients, the median delay increased from 7 (range 4.5-13) to 7.5 (range 5-13) weeks (P = 0.018). For MMN patients, the median delay between IVIg courses increased from 7 (range 5-12) to 9 (range 6-13) weeks (P = 0.011). There was no significant difference in median delay between IVIg courses for myasthenia gravis and LSS patients.

Table 4 summarises all results related to medical practices.

Clinical impact of IVIg treatment changes

Out of the 111 patients who had IVIg treatment changes, 99 were retained for this analysis; 12 were excluded due to lack of data and incomplete patient medical records. The ONLS, RODS and myasthenia muscle scores of these 99 patients showed that score deterioration occurred in 58, including 31 with moderate or clinically significant deterioration as defined in Methods. Among these 58 patients, 27 (47%) were classed in group 0 (low score deterioration), 21 (36%) in group 1 (moderate score deterioration), and 10 (17%) in group 2 (clinically significant deterioration). Eighteen (31%) patients had at least two types of IVIg treatment changes. For 17 (30%) patients, the clinical deterioration was directly linked to the shortage of IVIg. For six patients, only RODS deteriorated: these patients felt fatigue without an objective increase of deficit at clinical examination. Analysis of the functional score time course showed that IVIg treatment changes were significantly associated with clinical impact (P = 0.001).

Discussion

Growing indications for Ig in recent years has led to an increase in consumption. At the same time, lack of raw materials has led to Ig shortage problems. This study allowed us to evaluate the impact of this situation on patient care in a French university centre.

One particular interest of this study is to note that the context of shortage led to essential re-evaluations of IVIg treatments, particularly concerning doses and delay between IVIg courses. Indeed, 111 patients (78%) experienced one or more types of change to their IVIg regimen.

This study allowed us to observe an increase of delays between IVIg courses for CIDP, LSS, MMN and myasthenic patients when IVIg shortage developed. However, depending on patient response, compliance with a longer delay between IVIg courses was not always possible. This need to maintain short intervals between courses corresponds to the IVIgdependence phenomenon reported in several studies [START_REF] Elovaara | EFNS guidelines for the use of intravenous immunoglobulin in treatment of neurological diseases: EFNS task force on the use of intravenous immunoglobulin in treatment of neurological diseases[END_REF][START_REF] Phuong | The impacts of medication shortages on patient outcomes: a scoping review[END_REF].

Dose reductions were made in order to rationalise prescriptions. Analysing dose changes since 2016, we noted that myasthenia patients were affected the most by dose reductions. In our centre, medical practices tended to come closer to dose levels recommended by the French drug safety agency (Agence nationale de se ´curite ´du medicament, ANSM): 1 g/ kg. Indeed, a 2005 study compared 1 g/kg to 2 g/kg in the treatment of myasthenia gravis and revealed no significant difference in efficacy between the two doses [START_REF] Perez | Update on the use of immunoglobulin in human disease: A review of evidence[END_REF][START_REF] Eftimov | Intravenous immunoglobulin for chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Gajdos | Treatment of Myasthenia Gravis Exacerbation With Intravenous Immunoglobulin: A Randomized Double-blind Clinical Trial[END_REF].

It is more difficult to reduce IVIg doses in patients with stabilised disease [START_REF] Kuitwaard | Serum IgG levels in IV immunoglobulin treated chronic inflammatory demyelinating polyneuropathy[END_REF]. However, in a retrospective cohort study, gradual dose reduction was successful in 15 CIDP patients. Most patients began with an initial dose of 2 g/kg and were able to reduce this dose by about 63%. These results suggest that much smaller doses-at initiation or during r e v u e n e u r o l o g i q u e 1 7 8 ( 2 0 2 2 ) 6 1 6 -6 2 3 maintenance-could be effective, underlining the need for comparative tests [START_REF] Rajabally | Immunoglobulin G level variations in treated chronic inflammatory demyelinating polyneuropathy: clues for future treatment regimens?[END_REF].

The dose schedule and the dose adjustment strategy should both be adapted on a case-by-case basis, according to individual response. In the context of plasma shortage and regular lack of IVIg availability, dose reduction and wider treatment intervals lead to decreased IVIg consumption. An important issue in itself, wider treatment intervals could also involve improved patient quality-of-life and fewer hospitalisations.

This situation has also made it possible to discontinue certain IVIg treatments. IVIg treatments were stopped when patients were clinically stable, when complete symptom resolution was noted, or when there was no end-of-dose effect. As well, prescribers switched to corticosteroids, PE or an association of several immunomodulating treatments whenever possible in order to save Ig for patients who had no other alternatives, especially those with severe and chronic diseases [START_REF] Hughes | Randomized controlled trial of intravenous immunoglobulin versus oral prednisolone in chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Nobile-Orazio | Intravenous immunoglobulin versus intravenous methylprednisolone for chronic inflammatory demyelinating polyradiculoneuropathy: a randomised controlled trial[END_REF][START_REF] Latov | Timing and course of clinical response to intravenous immunoglobulin in chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Hughes | Intravenous immune globulin (10% caprylatechromatography purified) for the treatment of chronic inflammatory demyelinating polyradiculoneuropathy (ICE study): a randomised placebo-controlled trial[END_REF][START_REF] Van Schaik | Pulsed high-dose dexamethasone versus standard prednisolone treatment for chronic inflammatory demyelinating polyradiculoneuropathy (PREDICT study): a double-blind, randomised, controlled trial[END_REF][START_REF] Dyck | A plasma exchange versus immune globulin infusion trial in chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Hughes | Corticosteroids for chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Eftimov | Deterioration after corticosteroids in CIDP may be associated with pure focal demyelination pattern[END_REF][START_REF] Franques | Les polyradiculone ´vrites inflammatoires de ´mye ´linisantes chroniques[END_REF]. Albeit, it is known that patients with LSS can get worse after corticosteroid treatment [START_REF] Attarian | Response to treatment in patients with Lewis-Sumner syndrome[END_REF] and that MMN patients may have no other option than IVIg [START_REF] Pestronk | A treatable multifocal motor neuropathy with antibodies to GM1 ganglioside[END_REF][START_REF] Vlam | Multifocal motor neuropathy: diagnosis, pathogenesis and treatment strategies[END_REF][START_REF] Jinka | Treatment of multifocal motor neuropathy[END_REF][START_REF] Umapathi | Immunosuppressant and immunomodulatory treatments for multifocal motor neuropathy[END_REF]. The choice of treatment therefore has to be made on a case-bycase basis.

About 16% of patients, particularly those with myasthenia gravis or CIDP were switched to PE. Nevertheless, the technical aspects and potential complications of PE require considerable technical skill implying non-negligible training time for the medical team. Moreover, many teams prefer IVIg treatments because PE is a relatively invasive treatment. In addition, each PE session requires hospitalisation for a few hours and four to five sessions, every other day, are necessary for optimal clinical results similar to those obtained with four sessions of IVIg infusions [START_REF] Combe | Cou ˆt du traitement par e ´changes plasmatiques vs immunoglobulines intraveineuses en re ´animation[END_REF][START_REF] Hughes | Intravenous immunoglobulin for Guillain-Barre ´syndrome[END_REF][START_REF] Hughes | Corticosteroids for Guillain-Barre ´syndrome[END_REF].

Our study has some limitations. It was conducted in a single centre and was retrospective. Missing data hampered the inclusion of many patients. For this study, we analysed RODS, MMN-RODS, ONLS and myasthenia muscle scores that are recognised as fundamental and robust. As well, it would have been interesting to have evaluated fatigue and qualityof-life scores that were unfortunately unavailable for this retrospective analysis.

French guidelines, which recommended use of Ig by priority according to indications, made it possible to reevaluate treatment modalities, and thus to offer the most suitable treatment for each patient. This suggests that in the context of procurement constraints, a re-evaluation of the treatment regimen was conceivable for some patients. Such re-evaluations were effective in limiting the use of Ig in the context of national and international shortages. Nevertheless, this study demonstrated that treatment changes were not without side effects: we observed a deterioration of evaluation scores in about 31% of patients whose IVIg regimens were modified. This finding shows that IVIg treatment changes, directly related to IVIg shortages, had a negative impact on the clinical status of some patients. Indeed, the delay between IVIg courses is calculated according to the clinical deficit; an overly long interval can lead to disease flare-up, particularly for unstable patients. Moreover, for some diseases, IVIg appears to be the only treatment option. So, some patients without a real therapeutic alternative may have experienced therapeutic shortage. Moreover, maintenance treatments are not clearly defined; some practitioners reduce dose frequency and others dose level. Both techniques can induce deterioration. Practices need to be defined by scientific societies and justified in the literature.

A multidisciplinary consultation meeting is needed to initiate Ig treatment in CIDP and MMN patients. Furthermore, treatments for CIDP, MMN and myasthenia gravis cannot be initiated in France without prior consultation with a neuromuscular disease reference centre (La filie `re de sante ´maladies neuromusculaires, FILNEMUS). These meetings ensure optimal patient care and help regulate use of Ig. Regular evaluation of individual treatments is also part of this approach to improving appropriate use of Ig. In this context of procurement constraint, appropriate use of Ig has been a real public health issue and has required a change in practices and prescribing habits to reserve these products for warranted indications.

The interest of re-evaluating IVIg treatments remained significant in 2020. The COVID-19 pandemic has had a likely impact on global blood and plasma collection, on which Ig is dependent. This weakening of production due to a lack of raw material is likely to increase supply tensions in the next 6-10 months. The COVID-19 crisis heralds the beginning of a complicated period, and in this context, it can be expected that this type of study will develop more and more concerning drugs derived from blood or other products. As access to IVIg will be more difficult, other treatments should be considered to save plasma and Ig, for example rituximab or haematopoietic stem cell transplantation for second-line CIDP therapy should be discussed to spare IVIg [START_REF] Burt | Hematopoietic stem cell transplantation for chronic inflammatory demyelinating polyradiculoneuropathy[END_REF].

Conclusion

Our study showed a significant number of changes in IVIg treatments directly related to the shortage of IVIg: delay between IVIg courses, IVIg dose reduction and discontinuation of IVIg treatment during the study period. The context of shortage has made it necessary to reevaluate important treatment options. For most patients, changes in IVIg treatments have been maintained.

At an international level, shortage is also the main issue: the Food and Drug Administration works in close collaboration with the producers to reduce as much as possible tension in the Ig supply situation [START_REF] Vanhoutte | Rasch-built Overall Disability Scale for Multifocal motor neuropathy (MMN-RODS(#))[END_REF].

Treatment re-evaluations will continue to be important because of the fragility of the post-COVID market. Rationalising and harmonising at an international level would be interesting in order to save plasma in this shortage context as well as to move on to better use of Ig.

Standard protocol approvals, registrations, and patient consents

This study was approved by the institutional research ethics board, Assistance Publique-Ho ˆ pitaux de Marseille, (PADS20-246).

  Background. -Growing numbers of indications for intravenous immunoglobulins (IVIg) in recent years has resulted in an increase in the consumption of these products. A lack of raw material has led to IVIg shortage. The objective of this work was to evaluate the impact of this situation on patient care in one French referral centre considering practice modifications and clinical impact.Methods. -All patients treated with IVIg for chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy and myasthenia gravis from October 2017 to October 2018 were included.

3. 2 . 1 .

 21 Impact on patient care Out of 142 patients, 111 (78%) underwent a modification of their IVIg treatment. For the majority, changes were made at the height of the COVID-19 crisis; 45% of the modifications occurred in March 2018. Some patients had more than one type of IVIg treatment change during this period. Globally, we noted 147 IVIg treatment changes related to IVIg shortage. IVIg treatment was delayed in 75 (68%) patients, IVIg dose was decreased in 41 (37%), and IVIg treatment was discontinued in 31 (28%). Out of the 111 patients whose IVIg treatment was modified, 77 (69%) had one type of IVIg treatment change and 32 (29%) had two types of IVIg treatment changes; two patients (2%) had three types of IVIg treatment changes (first delay, then dose reduction, and finally complete discontinuation).

Fig. 1 -

 1 Fig. 1 -Study design.

r e v u e n e u r o l o g i q u e 1

 1

Table 2 -

 2 Distribution of IVIg treatment changes by dysimmune neuromuscular disease.

		Total of	IVIg treatment	IVIg	Discontinuation of	Number of patients with	Total of changes
		cases	delay	dose decrease	IVIg treatment	at least one change
	CIDP	47 (33%)	23 (49%)	12 (26%)	14 (30%)	35	49
	LSS	40 (28%)	23 (58%)	13 (33%)	2 (0.1%)	29	38
	MMN	39 (28%)	26 (67%)	10 (26%)	8 (21%)	33	44
	Myasthenia	16 (11%)	3 (19%)	7 (44%)	6 (38%)	14	16
	Total	142	75	41	31	111	147

Table 3 -

 3 Maintenance of IVIg treatment changes one year later after analysis period.

	N = 147 a	Modifications	Modifications not maintained
	IVIg Dose decrease	41	27 (66 %) b
	Delaying IVIg treatment	75	54 (72 %) c
	Stopping IVIg treatment	31	0
	Switch to plasma exchange	5	0
	Switch to corticosteroids	4	0
	Total	147 (45%)	81 (55%)

a We consider here 111 patients with modification corresponding to 147 modifications: 77 patients had 1 type of prescription changes, 32 patients had 2 types of prescription changes and 2 patients had 3 types. b Including 10 Ig interruptions. c Including 21 Ig interruptions.

Table 1 -

 1 Patient characteristics.

		r e v u e n e u r o l o g i q u e 1 7 8 ( 2 0 2 2 ) 6 1 6 -6 2 3
	n = 142	Value
	Age (years) AE IQR	61.5 AE 15.2
		Range = 26-86
	Weight (kg) AE IQR	76 AE 25
		Range = 38.5-173
	Sex ratio (H/F)	1.75
	Disease distribution
	CIDP	47 (33%)
	LSS	40 (28%)
	MMN	39 (28%)
	Myasthenia	16 (11%)
	Median immunoglobulin dose (g/kg)
	per indication AE IQR
	CIDP	1.67 AE 0.44
	LSS	1.71 AE 0.4
	MMN	1.69 AE 0.53
	Myasthenia	1.45 AE 0.6
	Median interval between doses (weeks)
	per indication AE IQR
	CIDP	7.5 AE 4
	LSS	8 AE 3
	MMN	8.25 AE 3.5
	Myasthenia	6 AE 2
	CIDP: chronic inflammatory demyelinating polyneuropathy; LSS:
	Lewis and Sumner syndrome; MMN: multifocal motor neuropathy.

Table 5

 5

	summarises features observed in all patients with
	deteriorating clinical scores.

Table 5 -

 5 Distribution of patients with deteriorating clinical score.

	Group 0	Group 1	Group 2	Total
	Low score	Moderate	Clinically significant
	deterioration	deterioration	deterioration

Criteria: Group 0: Same ONLS score, RODS score decrease < 4 points, myasthenia muscle score decrease < 5 points; Group 1: ONLS score increase ! 1 point; RODS score decrease ! 4 and < 6 points; myasthenia muscle score decrease ! 5 and < 10 points; Group 2: ONLS score increase ! 2 points, RODS score decrease ! 6 points and myasthenia muscle score decrease ! 10 points. * We consider here 3 types of IVIg treatment changes: IVIg treatment delay, discontinuation of IVIg treatment and IVIg dose decrease.

Table 4 -

 4 Evolution of median doses and median intervals between two IV Ig treatments, before and after the shortage depending on dysimmune neuromuscular disease.

	N = 51 a	Median doses (g/kg) AE IQR
		Before shortage-2016	After shortage-2019	P
	CIDP	1.68 AE 0.35	1.67 AE 0.3	0.722
	(n = 12)
	LSS	1.76 AE 0.25	1.72 AE 0.47	0.059
	(n = 16)
	MMN	1.83 AE 0.31	1.69 AE 0.49	0.034
	(n = 19)
	Myasthenia	1.73 AE 1	1.15 AE 0.5	0.423
	(n = 4)
	N = 51 a	Median intervals between treatments (weeks) AE IQR
		Before shortage-2016	After shortage-2019	P
	CIDP	7 AE 2	7.5 AE 4	0.011
	(n = 12)
	LSS	7.5 AE 2	8 AE 2.5	0.167
	(n = 16)
	MMN	7 AE 2	9 AE 3	0.018
	(n = 19)
	Myasthenia	5 AE 0.2	5.25 AE 0.5	0.5
	(n = 4)

a Patients who were not monitored during the period under consideration are excluded.

r e v u e n e u r o l o g i q u e 1 7 8 ( 2 0 2 2 ) 6 1 6 -6 2 3

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

All de-identified data and related documentation from this study are available upon request to qualified researchers without limit of time, subject to a standard data sharing agreement.

Disclosure of interest

The authors declare that they have no competing interest. r e f e r e n c e s


Manon Blin

Bruno Senghor

Jérôme Boissier

Stephen Mulero

Olivier Rey

Julien Portela

Development of environmental loop-mediated isothermal amplification (eLAMP) diagnostic tool for Bulinus truncatus field detection

Keywords: Environmental DNA, LAMP assay, Intermediate host control, Bulinus truncatus

Background Global changes are reshaping the distribution of vector-borne diseases by spreading vectors to previously non-endemic areas. Since 2013, urogenital schistosomiasis has emerged in Corsica and threatens European countries. Gastropod vectors release schistosome larvae that can infect humans who come into contact with freshwater bodies. Monitoring schistosomiasis host vectors is a prerequisite to understand and subsequently to control this pathogen transmission. Because malacological surveys are time consuming and require special expertise, the use of a simple molecular method is desirable. Methods The aim of this study is to develop a ready-to-use protocol using the LAMP (loop-mediated isothermal amplification) method to detect environmental DNA of Bulinus truncatus, vector of Schistosoma haematobium. Interestingly, LAMP method possesses all the characteristics required for adaptability to field conditions particularly in low-income countries: speed, simplicity, lyophilized reagents, low cost and robustness against DNA amplification inhibitors. We have tested this new method on Corsican water samples previously analysed by qPCR and ddPCR.

Results

We demonstrate that our diagnostic tool B. truncatus eLAMP (Bt-eLAMP) can detect the eDNA of Bulinus truncatus as effectively as the two other methods. Bt-eLAMP can even detect 1/4 of positive samples not detectable by qPCR. Moreover, the complete Bt-eLAMP protocol (sampling, sample preprocess, amplification and revelation) does not require sophisticated equipment and can be done in 1 ½ h. Conclusions LAMP detection of environmental DNA provides large-scale sensitive surveillance of urogenital schistosomiasis possible by identifying potentially threatened areas. More generally, eLAMP method has great potential in vector-borne diseases and ecology.

Background

It is well established that global changes increase the risk of infectious disease outbreaks [START_REF] Ni | Emerging and neglected infectious diseases: insights, advances, and challenges[END_REF][START_REF] Baker | Infectious disease in an era of global change[END_REF][START_REF] Tredennick | Anticipating infectious disease re-emergence and elimination: a test of early warning signals using empirically based models[END_REF]. The successive epidemic waves over the past decade such as the swine flu pandemic, numerous Ebola virus outbreaks in West Africa, 2015 Zika virus outbreak and the COVID-19 pandemic all resulted in significant mortality and morbidity while spreading to multiple countries [START_REF] Baker | Infectious disease in an era of global change[END_REF]. The incidences of vectorborne diseases in humans and animals are impacted by climate change and human lifestyle (e.g. migration, pollution, urbanization) [START_REF] Ni | Emerging and neglected infectious diseases: insights, advances, and challenges[END_REF][START_REF] Caminade | Impact of recent and future climate change on vector-borne diseases[END_REF][START_REF] Allen | Global hotspots and correlates of emerging zoonotic diseases[END_REF]. For several years, many of them have been emerging or re-emerging worldwide. For example, the geographical range of tickborne encephalitis virus and the human incidence are increasing throughout Europe, including France, in areas that were previously non-endemic [START_REF] Gonzalez | A one-health approach to investigating an outbreak of alimentary tickborne encephalitis in a non-endemic area in France (Ain, Eastern France): a longitudinal serological study in livestock, detection in ticks, and the first tick-borne encephalitis virus isolation and molecular characterisation[END_REF]. Although schistosomiasis is a disease linked to poverty and lack of potable water in households, schistosome infection has been identified in southern Europe (France and Spain) [START_REF] Salas-Coronas | Evidence of autochthonous transmission of urinary schistosomiasis in Almeria (southeast Spain): an outbreak analysis[END_REF][START_REF] Boissier | Schistosomiasis reaches Europe[END_REF]. Gastropod vectors release schistosome larvae that can infect humans who come into contact with freshwater bodies. In summer 2013, > 106 cases were diagnosed in Corsica, a French Mediterranean island known for its touristic attractiveness [START_REF] Boissier | Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study[END_REF][START_REF] Noel | Large outbreak of urogenital schistosomiasis acquired in Southern Corsica, France: monitoring early signs of endemicization?[END_REF]. Since then, a few cases of autochthonous transmission have persisted year after year [START_REF] Berry | Evidence for a permanent presence of schistosomiasis in Corsica, France[END_REF]. For many parasitic diseases, global changes lead to a spatial redistribution of the vectors or the intermediate hosts which transmit the diseases and may also lead to disease emergence [START_REF] El-Sayed | Climatic changes and their role in emergence and re-emergence of diseases[END_REF][START_REF] Giron | Review of Arbovirus surveillance in 2019: transition to surveillance for confirmed cases of Chikungunya, Dengue and Zika Virus in metropolitant France[END_REF][START_REF] Musso | Zika virus[END_REF][START_REF] Mysterud | Contrasting emergence of Lyme disease across ecosystems[END_REF][START_REF] Millins | Emergence of Lyme disease on Treeless Islands, Scotland, United Kingdom[END_REF]. Mapping infectious disease outbreak areas is essential to both identify populations at risk of infection and communicate and warn the public [START_REF] Kraemer | Progress and challenges in infectious disease cartography[END_REF]. Concerning vectorborne diseases, since the transmission is dependent on the presence of the vectors, mapping their spatial distributions is necessary for disease control. It has been shown in several African countries that snail control campaigns play a major role in controlling snail-borne diseases (SBDs) and that regular application of molluscicides probably contributes to the elimination of the SBDs in at-risk areas [START_REF] Rollinson | Time to set the agenda for schistosomiasis elimination[END_REF][START_REF] King | Systematic review and meta-analysis of the impact of chemicalbased Mollusciciding for control of Schistosoma mansoni and S. haematobium transmission[END_REF][START_REF] Sokolow | Global assessment of schistosomiasis control over the past century shows targeting the snail intermediate host works best[END_REF]. However, the monitoring of SBDs, particularly those transmitted via freshwater snail species, is less obvious. The spatial and temporal heterogeneity of the distribution of freshwater molluscs explains the difficulty of establishing these risk maps [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF][START_REF] Lamy | Testing metapopulation dynamics using genetic, demographic and ecological data[END_REF]. Conventional sampling methods are therefore laborious and time-consuming, and they require malacologists for taxonomic identification, which have become increasingly scarce in recent years as molecular biology has taken off. Therefore, it is currently very difficult to monitor snail populations on a large spatial and temporal scale [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF][START_REF] Lamy | Testing metapopulation dynamics using genetic, demographic and ecological data[END_REF].

The development of tools to simplify the monitoring of SBDs has been considered to address this issue, based on environmental DNA (eDNA) [START_REF] Kamel | Detecting and identifying Schistosoma infections in snails and aquatic habitats: a systematic review[END_REF]. eDNA amplification tmethods were developed to detect many parasite vectors such as Aedes albopictus [START_REF] Schneider | Detection of invasive mosquito vectors using environmental DNA (eDNA) from water samples[END_REF] and also snail hosts such as Galba truncatula and Austropeplea tomentosa [START_REF] Jones | Detection of Galba truncatula, Fasciola hepatica and Calicophoron daubneyi environmental DNA within water sources on pasture land, a future tool for fluke control?[END_REF][START_REF] Rathinasamy | Development of a multiplex quantitative PCR assay for detection and quantification of DNA from Fasciola hepatica and the intermediate snail host, Austropeplea tomentosa, in water samples[END_REF], both species being involved in the transmission of the liver fluke (Fasciola hepatica). The detection of eDNA is usually done using quantitative PCR DNA amplification technique. More recently, the eDNA detection of Oncomelania hupensis and Bulinus truncatus, involved in the transmission of Schistosoma japonicum and Schistosoma haematobium, respectively, has been made possible by either quantitative PCR (qPCR) or digital droplet PCR (ddPCR) [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF][START_REF] Calata | Analysis of environmental DNA and edaphic factors for the detection of the snail intermediate host Oncomelania hupensis quadrasi[END_REF]. The results obtained in these last studies showed high-sensitivity and -specificity detection. Detection of B. truncatus eDNA by ddPCR allowed finding up to 0.06 copies/μl [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF]. This represents a significant advance in the development of tools to simplify SBD monitoring. However, all these techniques require sophisticated and expensive equipment for both sample preparation (i.e. the use of expensive and time-consuming DNA extraction kits to obtain eDNA without PCR inhibitors) and amplification equipment (i.e. LightCycler or ddPCR). These costly techniques require a well equipped laboratory, which is rarely available in schistosome-endemic areas. Development of cost-effective and field-adapted tools for realization of snail distribution maps is still needed [START_REF] Kraemer | Progress and challenges in infectious disease cartography[END_REF][START_REF] Kamel | Detecting and identifying Schistosoma infections in snails and aquatic habitats: a systematic review[END_REF][START_REF] Kincaid-Smith | Emerging Schistosomiasis in Europe: a need to quantify the risks[END_REF]. The loopmediated isothermal amplification (LAMP) technique has the potential to make this possible [START_REF] Mori | Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[END_REF][START_REF] Notomi | Loop-mediated isothermal amplification of DNA[END_REF][START_REF] Notomi | Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects[END_REF]. Interestingly, the LAMP method possesses all the characteristics required for adaptability to field conditions particularly in lowincome countries: speed, simplicity, lyophilized reagents, low cost and robustness against DNA amplification inhibitors [START_REF] Mori | Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[END_REF][START_REF] Notomi | Loop-mediated isothermal amplification of DNA[END_REF][START_REF] Kumar | Advanced lyophilised loop mediated isothermal amplification (L-LAMP) based point of care technique for the detection of dengue virus[END_REF]. Indeed, using a Bst polymerase enzyme with strand displacement activity and four to six primers hybridizing on six to eight regions of the target sequence, the exponential DNA amplification reaction can be performed at constant temperature, without prior denaturation of the DNA double strands. Indeed, the hybridization of the primers will create stem-loop structures on the newly amplified strands leading to the exponential multiplication of the number of new amplification start zones. We finally obtain a smear composed of several fragments of varied sizes which form a ladder after revelation by electrophoresis gel. Thus, the reaction is performed very quickly (usually < 1 h) at a constant temperature of around 63 °C in a simple heating block [START_REF] Mori | Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[END_REF][START_REF] Notomi | Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects[END_REF][START_REF] Kumar | Isothermal amplification-based methods for assessment of microbiological safety and authenticity of meat and meat products[END_REF]. Since no large or expensive equipment is required to perform a LAMP, recent studies have focused on the development of portable devices to perform field diagnosis as close as possible to the sampling site [START_REF] Buultjens | Low-cost, opensource device for high-performance fluorescence detection of isothermal nucleic acid amplification reactions[END_REF][START_REF] Velders | T-cup: a cheap, rapid, and simple home device for isothermal nucleic acid amplification[END_REF][START_REF] Chen | A portable multi-channel turbidity system for rapid detection of pathogens by loop-mediated isothermal amplification[END_REF][START_REF] Matute | Homebrew reagents for low cost RT-LAMP[END_REF]. Due to its robustness against inhibitors, it can be performed on samples with very simplified preparation, without the need for DNA extraction kits to purify the DNA, which can also be done in the field [START_REF] Detemmerman | Innovative PCR without DNA extraction for African sickle cell disease diagnosis[END_REF][START_REF] Gandasegui | The rapid-heat LAMPellet method: a potential diagnostic method for human urogenital Schistosomiasis[END_REF]. DNA amplification can be detected by naked-eye visualization, gel electrophoresis or real-time fluorescence, offering flexibility of use in field settings [START_REF] Buultjens | Low-cost, opensource device for high-performance fluorescence detection of isothermal nucleic acid amplification reactions[END_REF][START_REF] Chen | A portable multi-channel turbidity system for rapid detection of pathogens by loop-mediated isothermal amplification[END_REF]. Recent studies have shown that it is possible to detect eDNA by LAMP [START_REF] Lee | Rapid and in-situ detection of fecal indicator bacteria in water using simple DNA extraction and portable loop-mediated isothermal amplification (LAMP) PCR methods[END_REF][START_REF] Davis | Rapid detection of Galba truncatula in water sources on pasture-land using loop-mediated isothermal amplification for control of trematode infections[END_REF][START_REF] Williams | Isothermal amplification of environmental DNA (eDNA) for direct fieldbased monitoring and laboratory confirmation of Dreissena sp[END_REF][START_REF] Feist | Genetic detection of freshwater harmful algal blooms: a review focused on the use of environmental DNA (eDNA) in Microcystis aeruginosa and Prymnesium parvum[END_REF]. An environmental LAMP (eLAMP) was developed to detect the presence of faecal indicator bacteria in water in 1 h, without sophisticated laboratory equipment or highly trained personnel [START_REF] Lee | Rapid and in-situ detection of fecal indicator bacteria in water using simple DNA extraction and portable loop-mediated isothermal amplification (LAMP) PCR methods[END_REF]. Two teams succeeded in amplifying mollusc eDNA (Galba truncatula and Dreissena sp.) from water samples with LAMP [START_REF] Davis | Rapid detection of Galba truncatula in water sources on pasture-land using loop-mediated isothermal amplification for control of trematode infections[END_REF][START_REF] Williams | Isothermal amplification of environmental DNA (eDNA) for direct fieldbased monitoring and laboratory confirmation of Dreissena sp[END_REF].

The aim of this study is to develop a rapid diagnostic LAMP tool to detect the environmental DNA of Bulinus truncatus, intermediate host of Schistosoma haematobium. Schistosomiasis is the second most common parasitic disease after malaria and remains a neglected tropical disease. Despite its high incidence (230 million infected people and 200,000 deaths per year [START_REF]Prevention and control of schistosomiasis and soil-transmitted helminthiasis : report of a WHO expert committee[END_REF]), efforts to conduct snail control campaigns are very low [START_REF] Kincaid-Smith | Emerging Schistosomiasis in Europe: a need to quantify the risks[END_REF][START_REF] Stensgaard | Schistosomes, snails and climate change: current trends and future expectations[END_REF][START_REF]Global vector control response: an integrated approach for the control of vector-borne diseases[END_REF]. However, in 2017, WHO called for refocusing on snail control to sustain progress, calling on member states to develop or adapt national vector control strategies, aiming to reduce the incidence of vector-borne diseases, including schistosomiasis, by at least 40% by 2025 [START_REF]Global vector control response: an integrated approach for the control of vector-borne diseases[END_REF]. Thus, cost-effective, field-appropriate tools for rapid mapping of snail distribution are needed for this disease [START_REF] Kamel | Detecting and identifying Schistosoma infections in snails and aquatic habitats: a systematic review[END_REF]. Compared to qPCR or ddPCR amplification methods, the eLAMP seems advantageous for field applicability in low-income countries. However, this advantage disappears if the DNA extraction protocol that precedes amplification is not adapted to field conditions like using DNA extraction kits. Our work consisted in (i) development of B. truncatus-specific eLAMP assay, (ii) comparison of the results obtained by LAMP on an eDNA bank with those obtained by qPCR and ddPCR and (iii) development of a full field-friendly protocol of detection.

Methods

Material

The Bulinus truncatus eLAMP (Bt-eLAMP) diagnostic tool was developed from both an environmental DNA (eDNA) bank and newly extracted DNA from molluscs.

The eDNA bank is composed of 200 samples previously collected on Corsica island, France [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF]. These samples were DNA extracted from water filtration membranes collected in two distinct rivers from southern Corsica. Urogenital schistosomiasis transmission has recently been evidenced in these two rivers [START_REF] Boissier | Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study[END_REF][START_REF] Noel | Large outbreak of urogenital schistosomiasis acquired in Southern Corsica, France: monitoring early signs of endemicization?[END_REF][START_REF] Berry | Schistosomiasis haematobium, Corsica[END_REF]. Three different volumes of water (1, 3 and 5 l) from both the shore and the streambeds of the rivers were collected. Four sites were sampled in the Cavu River: the Mulinu bridge and Tyroliana park sites (sampled in duplicate), the three pools site where B. truncatus are present and the water intake site where B. truncatus are absent. A single site was sampled from the Solenzara River where B. truncatus are present. Negative controls were made by filtering pure spring water along the river. In addition to these sampling sites in Corsica, eDNA was also sampled from an artificial channel within the campus of Perpignan University, where B. truncatus has never been recorded, as a negative control. The eDNA extraction protocol from the resulting water filtration membranes was previously described [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF] and is briefly recalled here: each filtration membrane was divided into four quarters and extracted using DNeasy ® Blood & Tissue Kit (QIAGEN). From the 200 samples, 144 eDNA samples were collected where B. truncatus were present and 56 eDNA samples collected where B. truncatus were not present, and they were amplified using qPCR. A sample was considered positive if at least one quarter membrane was positive. The ddPCR reactions were run on pools of DNA extracts from the four quarters of each membrane (i.e. 50 reactions) [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF]. Results per membrane obtained with both qPCR and ddPCR were compared to eLAMP (present study). Results per membrane quarter obtained by qPCR were also compared to eLAMP.

In addition to eDNAs, and to test the specificity of our primer sets, DNA from freshwater snails (Bulinus sp. And other phylogenetically related genera) was also extracted using the DNeasy ® Blood & Tissue Kit (QIAGEN) following the tissue extraction protocol. Briefly, after grinding each snail, the samples were put in a Speedvac™ DNA 130 (Savant™, Thermo Scientific™) at 65 °C for 20 min to remove any alcohol or water residuals. The snails were then lysed at 56 °C for 4 h in a solution containing 20 ml proteinase K and 180 ml ATL buffer. The remaining steps were performed according to the manufacturer's instructions. The DNA concentration was measured using a Qubit ® 2.0 fluorometer. These last DNAs were diluted with ultrapure water to obtain final concentrations of 0.5 ng/μl. The resulting DNA extracts and dilutions were then stored at -20 °C until used.

eLAMP primer set design

The DNA target selection was exhaustive to maximize the chance of success in the development of the eLAMP diagnostic tool. The ITS2; 5,8S; 18S; COI; and 28S B truncatus DNA regions were downloaded from GenBank database (NCBI, https:// www. ncbi. nlm. nih. gov/ genba nk/; accession numbers: MH361757 for 5,8S; KJ157340 for 18S [START_REF] Zein-Eddine | Phylogeny of seven Bulinus species originating from endemic areas in three African countries, in relation to the human blood fluke Schistosoma haematobium[END_REF]; KJ157370 for 28S [START_REF] Zein-Eddine | Phylogeny of seven Bulinus species originating from endemic areas in three African countries, in relation to the human blood fluke Schistosoma haematobium[END_REF]; MT707426 for COI [START_REF] Chibwana | Assessing the diversity and distribution of potential intermediate hosts snails for urogenital schistosomiasis: Bulinus spp. (Gastropoda: Planorbidae) of Lake Victoria[END_REF] and MG757890 for ITS2 [START_REF] Abe | Phylogeography of Bulinus truncatus (Audouin, 1827) (Gastropoda: Planorbidae) in selected African Countries[END_REF]). When available, sequences from other Bulinus species were downloaded from GenBank database to develop a B. truncatus-specific detection tool. The design of primer sets was carried out using Primer Explorer V5 software (Eiken Chemical Co., Ltd., Japan; http:// prime rexpl orer. jp/e/) following the criteria described in "A Guide to LAMP primer designing" (https:// prime rexpl orer. jp/e/ v5_ manual/index. html). The sets had to meet the following parameters: length of each primer between 18 and 22 bp; Tm between 64 and 67 °C for F1c/B1c pair; Tm between 59 and 62 °C for F2/B2 and F3/B3 pairs; GC rate between 50 and 65%; dG threshold < -4 kcal/mol for 5' and 3' stability and between 0 and -1 kcal/mol for dimer check. Primer dimer estimation and stability of each primer set were checked online using the "Multiple Primer Analyzer" (Thermo Scientific Web Tool) and OligoAnalyzer™ Tool (IDT™). All the primers selected were synthesized by Eurogentec (Seraing, Belgium).

LAMP reaction

For each designed primer set several amplification assays were performed by adjusting the amplification reaction parameters (i.e. MgSO4, primers and betaine concentration, reaction time and temperature). The selected protocol was as follows: a total volume of 10 μl containing 1.2 μM of the internal primers FIP and BIP, 0.2 μM of the external primers F3 and B3, 0.4 μM of the LOOP primers LB and LF, 1.0 mM of each dNTP (New England Biolabs), 1X Isothermal Amplification Buffer II reaction buffer [20 mM Tris-HCl, 10 mM (NH4)2SO4, 150 mM KCl, 2 mM MgSO4, 0. 1% Tween ® 20, pH 8.8@25 °C (New England Biolabs, UK)], 3 mM additional MgSO4 (New England Biolabs, UK), 1.2 M betaine and 1 U of Bst 2.0 WarmStart DNA polymerase (New England Biolabs, UK) along with 2.5 μl DNA template. Reaction tubes were placed in a battery-operated heating block at 63 C for 45 min followed by an enzyme inactivation phase at 80 °C for 5 min if the results were not visualised immediately after the reaction. Result visualisations were done according to different protocols classically used in LAMP DNA amplification: (i) final point visual detection of fluorescence after adding 2 μl of 1:50 diluted 10,000× concentration of SYBR Green (Invitrogen) (green: positive; orange: negative); (ii) final point visual detection of the LAMP products on 2% agarose gel electrophoresis with 2 μl MIDORI Green Advance (Nippon Genetics) for 50 ml agarose gel, revealed by UVs; (iii) real-time detection by incorporating Sybr-Green 0.25× in the reaction mix and monitoring the amplification in a Genie III portable device (Optigene Ltd).

Specificity and limit of detection

The specificity of all designed primer sets was assessed on DNA templates from 12 freshwater snail species ( 1) were evaluated to visualise a possible intraspecific variability. Only the primer set amplifying exclusively B. truncatus DNA was kept for the ensuing experiments.

The limit of detection (LOD) was assessed by LAMP assays on DNA from B. truncatus tenfold serially diluted from 10 ng/μl to 1.10-9 ng/μl before and after adding LOOP primers to the reaction. From the detection limit DNA concentration obtained, the number of B. truncatus target copies per microlitre was estimated using the following equation:

where Nc = the number of B. truncatus target copies per microlitre; C = limit detection DNA concentration in ng/ μl; V = volume (here, 1 μl); M = average molar mass of a nitrogenous base = 309 g/mol; Sg = B. truncatus genome size = 1.2.109 pb; NA = Avogadro number; NrDNA = average rDNA copy number in a genome = 93 copies [START_REF] Sochorova | Evolutionary trends in animal ribosomal DNA loci: introduction to a new online database[END_REF][START_REF] Gregory | Eukaryotic genome size database[END_REF][START_REF] Prokopowich | The correlation between rDNA copy number and genome size in eukaryotes[END_REF][START_REF] Iwanaga | Variations in ribosomal DNA copy numbers in a genome of Trichophyton interdigitale[END_REF][START_REF] Malone | Balancing copy number in ribosomal DNA[END_REF][START_REF] Johnson | Determination of ribosomal DNA copy number and comparison among strains of Coccidioides[END_REF].

Field applicability

We have developed a new field-friendly eDNA concentration protocol allowing us to take full advantage of the field benefits of LAMP. The water is collected in a clean plastic commercial bottle on the surface of the water body. Then, 500 ml to 1500 ml of water is filtered along the river using a 0.45 μm filtration unit (PES VWR 1L 514-0301) and a manual pump. The water is filtered until the membrane is blocked because of sediment stacking. The membrane is cut in four quarters with a scalpel blade and deposited in a single 25-ml Falcon™ tube containing 15 ml lysis buffer [100 mM NaCl, 250 mM EDTA, 5% SDS and 10 mM Tris-HCl (pH = 8)]. Clamps used to remove the membranes from filtration units are decontaminated before each sampling by successively placing them in a 10% bleach bath for 15 s and then 15 s in a DNA AWAY™ solution (Thermo Scientific). After vigorous agitation, the liquid is collected with a 20-ml syringe. The liquid is pre-filtered with a 40-μm membrane to remove the larger sediments and then filtered with a 0.45-μm (13-mm) Whatman ® GD/XP syringe filter (PTFE Sigma Aldrich WHA69742504). The last filter is washed with 5 ml distilled water and dried with the same volume of air. The eDNA is finally recovered by reverse filtration with 500 μl ultrapure water thanks to a syringe coupled with a conical 18 GA dispensing needle (Metcal).

This last protocol was assessed in two Senegalese water bodies investigated in July 2022. The Lampsar site (16°06′29′′N; 16°20′59′′W) and Diama site (16°12′36′′N; 16°24′10′′W) are both situated along the Senegal River in Saint-Louis region, Senegal. At each site, water samples were collected in triplicate with about 10 m between samplings. Seven hundred millilitres, two times 500 ml and three times 500 ml were collected for Lampsar and Diama sites, respectively. At each site negative controls were carried out by filtering 1500 ml pure spring water strictly following the same procedure as for biological samples. The presence on site of B. truncatus was verified by sampling molluscs for 1 h by two malacologists.

Statistical analysis

The agreement between results obtained with the newly developed LAMP and the qPCR for the detection of B. truncatus DNA on the 200 field eDNA library were statistically analysed calculating a kappa coefficient analysis using psych package (version 2.2.9) in R software. Field sensitivities on the eDNA bank were calculated with a 95% confidence interval using stats package (version 3.6.2) in R software

Results

Specificity and limit of detection of selected LAMP primer sets

A total of 18 primer sets were designed. After parameter adjustment and sensitivity and specificity assessment, we have selected the best candidate which targets the ITS2" marker (Table 2). An additional file shows the primer alignment on the ITS2 sequence (see Additional file 1). Primer dimer minimum dG for this set was -0.93 kcal/ mol. Bt-eLAMP specificity was assessed by testing extracted DNA from 11 non-targeted species and five targeted molluscs from different localities. Figure 1a shows that only the five B. truncatus DNA samples are amplified. Sensitivity test (Fig. 1b) shows that the LOD is 1 fg/μl (i.e. equivalent to about Nc = 0.07 copies per microlitre) with complete primer set (including LOOP primers). The detection limit was 1 pg/μl before adding the LOOP primers.

Comparison of LAMP to qPCR and ddPCR amplification methods on eDNA samples

Figure 2 shows detection accuracy comparison among LAMP, qPCR and ddPCR amplification methods on eDNA samples collected from two Corsican rivers. The Bt-eLAMP method provided the same results as those obtained using the qPCR and ddPCR. All membranes are positive except for the "1LStreambed" membrane from the Mulinu bridge two site. No positive signal was detected from the six field negative controls or from the University site where B. truncatus never occurred or for samples collected at Water intake site from the Cavu River where no B. truncatus were collected (Fig. 2). Considering results obtained per membrane quarter where B. truncatus was present, 110 of 144 samples were successfully amplified with LAMP; meanwhile, 91 of 144 samples were successfully amplified with qPCR. The sensitivities of LAMP and qPCR methods were estimated to be 76.4% (95% CI 68.6-83.1) and 63.2% (95% CI 54.8-71.1), respectively. Kappa agreement obtained with these results is equal to 0.51 (Table 3).

Field applicability

The use of LAMP amplification as field-friendly detection method was improved by developing a new eDNA concentration protocol that could be done along the water bodies. The entire protocol was tested on Lampsar and Diama sites, Senegal. In these two sites, respectively, 95 and 229 B. truncatus were sampled by two malacologists for 1 h sampling. Four of the 95 B. truncatus sampled from Lampsar site emitted cercariae after 2 h light exposure. Bulinus globosus was present at both sites. Bulinus forskalii was only present at Diama site, while we found Biomphalaria pfeifferi only at Lampsar site. The three eDNA samplings from the two sites were positive (Fig. 3) showing 100% accuracy of the method. None of the negative controls were positive. Samples of B. truncatus eDNA were amplified on average 12 min after amplification of the positive controls (B. truncatus extracted DNA).

Discussion

Global changes are reshaping the distribution of vector-borne diseases by spreading vectors to previously non-endemic areas. Therefore, the development of costeffective and field-adapted tools for vector distribution maps is necessary [START_REF] Kraemer | Progress and challenges in infectious disease cartography[END_REF][START_REF] Kincaid-Smith | Emerging Schistosomiasis in Europe: a need to quantify the risks[END_REF]. In the present study, we have developed a rapid LAMP diagnostic tool to detect the environmental DNA of a freshwater snail vector (i.e. B. truncatus) of a tropical parasite (i.e. S. haematobium). Our Bt-eLAMP successfully amplified an ITS2 DNA marker fraction of B. truncatus after 45 min of incubation at 63 °C. In vitro assays evidenced the high sensitivity of the Bt-eLAMP with a LOD equal to 1 fg/μl when it included LOOP primers. Considering that the LOD was 1 pg/μl before adding the LOOP primers, we deduce that the addition of LOOP primers allows a gain in sensitivity of a factor of 1000. From a methodological point of view, this highlights the importance of designing primer sets allowing inclusion of LOOP primers. The detection limits of the developed LAMP techniques are generally between 100-0.1 fg/μl [START_REF] Feist | Genetic detection of freshwater harmful algal blooms: a review focused on the use of environmental DNA (eDNA) in Microcystis aeruginosa and Prymnesium parvum[END_REF][START_REF] Abbasi | Detection of Schistosoma mansoni and Schistosoma haematobium DNA by Loop-mediated isothermal amplification: identification of infected snails from early prepatency[END_REF][START_REF] Gandasegui | Biompha-LAMP: a new rapid loop-mediated isothermal amplification assay for detecting Schistosoma mansoni in Biomphalaria glabrata snail host[END_REF][START_REF] Caldeira | Use of molecular methods for the rapid mass detection of Schistosoma mansoni (Platyhelminthes: Trematoda) in Biomphalaria spp. (Gastropoda: Planorbidae)[END_REF]. Our LOD is lower compared to the LOD previously evaluated for the LAMP eDNA detection of the freshwater snail vector of fasciolosis (Galba truncatula) [START_REF] Davis | Rapid detection of Galba truncatula in water sources on pasture-land using loop-mediated isothermal amplification for control of trematode infections[END_REF]. These last authors measured a 0.349 pg/μl LOD [START_REF] Davis | Rapid detection of Galba truncatula in water sources on pasture-land using loop-mediated isothermal amplification for control of trematode infections[END_REF]. When estimating the copy number equivalence (0.07 copies per microlitre) with the complete primer set (i.e. including LOOP primers), we found a sensitivity close to that obtained using ddPCR (0.06 copies per microlitre) [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF]. Our Bt-eLAMP showed equal sensitivity compared to the ddPCR, which is considered the most sensitive DNA amplification technique of all [START_REF] Falzone | Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection[END_REF][START_REF] Kojabad | Droplet digital PCR of viral DNA/RNA, current progress, challenges, and future perspectives[END_REF].

Our Bt-eLAMP assay showed 100% specificity for B. truncatus even considering closely related snail species. This suggests that the Bt-eLAMP assay is robust enough to detect B. truncatus eDNA in areas where other snails co-occur without risk of cross-reactivity. Our tool can also allow species determination between B. truncatus and other Bulinus species such as B. globosus, which are morphologically indistinguishable. On the other hand, the usefulness of this high specificity could be questioned if we consider that some molluscs' sister species, such as B. globosus, could also be involved in the transmission of urogenital schistosomiasis [START_REF] Catalano | Multihost transmission of Schistosoma mansoni in Senegal, 2015-2018[END_REF]. It would be interesting to also develop eDNA diagnostic tools allowing a pan-specific detection of both B. truncatus and B. globosus or all species of the Bulinus genus. The difficulty will be to find an equilibrium between sufficiently conserved regions between Bulinus species and sufficiently different from other gastropods to obtain a genus-specific primer set. This last task is more difficult as the number of primers required in LAMP and the positioning constraints between them on the target sequence increase the length of the sequence to be found meeting these requirements.

Considering our eDNA bank, the Bt-eLAMP method provided exactly the same overall detection capacity compared to the qPCR or the ddPCR [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF]. Considering each membrane quarter, with 76.4% (95% CI 68.6-83.1) and 63.2% (95% CI 54.8-71.1) of positive detection using LAMP or qPCR, respectively, the LAMP method showed higher detection capacity compared to qPCR [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF]. The kappa coefficient highlights moderate agreement between the two methods. Fifteen membrane quarters are negative in LAMP while they are positive in qPCR; conversely, 34 membrane quarters are negative in qPCR and positive in LAMP. The presence of PCR inhibitors could explain these last results. Indeed, it has already been proven that, in contrast to qPCR, LAMP is robust against inhibitors [START_REF] Mori | Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[END_REF][START_REF] Kaneko | Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances[END_REF].

Development of field friendly amplification methods such as LAMP need to be accompanied by the development of a field-friendly pre-process of the sample. Use of DNA extraction kits that have a low yield due to their high purification level requires special apparatuses such as centrifuges, which are costly. Therefore, we have developed a new field-friendly water sample pre-process to concentrate the eDNA. The sampling and pre-process protocol were done in 45 min in the field without electricity, which represents a significant advance in SBDs monitoring. Nevertheless, several improvements can be made to our protocol, especially on the water filtration step. The manual pump used limits the speed and convenience of the protocol since it took 30 min per sample to filter just 500 ml of water with difficulties. A 12-V battery-powered diaphragm water pump coupled with an adapted filter capsule was used to filter up to 50 l water in a DNA survey targeting trematodes in water [START_REF] Douchet | Make visible the invisible: optimized development of an environmental DNA metabarcoding tool for the characterization of trematode parasitic communities[END_REF]. To further enhance the practicability and speed of the BteLAMP assay, an improved battery-operated pump could replace the manual pump. The compatibility between a motorised pump and the filter units used in our protocol needs to be tested.

We used the Genie III to obtain our field results from Senegal, which is a useful, easily transportable, batteryoperated field tool, allowing visualization of reaction kinetics. Coupled with freeze-dried reagents, this type of portable device makes it possible to carry out the entire protocol (sampling, pre-process, amplification and revelation) in the field [START_REF] Kumar | Advanced lyophilised loop mediated isothermal amplification (L-LAMP) based point of care technique for the detection of dengue virus[END_REF]. We should use another portable device for low-income countries where the use of the Genie III may be a financial obstacle. The COVID-19 global pandemic involved the development of several tools to perform low-cost and field-friendly LAMPs [START_REF] Buultjens | Low-cost, opensource device for high-performance fluorescence detection of isothermal nucleic acid amplification reactions[END_REF][START_REF] Velders | T-cup: a cheap, rapid, and simple home device for isothermal nucleic acid amplification[END_REF][START_REF] Rezaei | A portable RT-LAMP/CRISPR machine for rapid COVID-19 screening[END_REF][START_REF] Sreejith | A portable device for LAMP based detection of SARS-CoV-2[END_REF][START_REF] Choi | An ultracompact real-time fluorescence loopmediated isothermal amplification (LAMP) analyzer[END_REF]. A low-cost and open-source LAMP diagnostic device has been developed that allows realtime amplification and fluorescence reading in the same way as the Genie III [START_REF] Buultjens | Low-cost, opensource device for high-performance fluorescence detection of isothermal nucleic acid amplification reactions[END_REF]. It has even been proven that the LAMP reaction can be done using a coffee capsule filled with phase change material, called a "T-cup", allowing incubation at 63 °C in boiling water and a nakedeye revelation [START_REF] Velders | T-cup: a cheap, rapid, and simple home device for isothermal nucleic acid amplification[END_REF]. In another study, the revelation step of a smartphone-based LAMP could be assessed with a smartphone application coupled with filters allowing the fluorescence reading. In the future, the current LAMP test should be improved with freeze-dried reagents and this type of device and then tested on a large sampling.

Conclusions

In this study, we developed a full field-friendly LAMP assay detecting the presence of the trematode intermediate snail host B. truncatus DNA in the environment. Due to LAMP's advantages of being fieldfriendly and allowing cost-effective pre-process samples, Bt-eLAMP test may be a valuable new tool for quickly and efficiently establishing recurrent risk maps for urogenital schistosomiasis. eLAMP has also been used to detect the presence of intestinal parasites in wastewater during treatment, even offering the possibility of looking for Schistosoma sp. eDNA directly [START_REF] Ajonina | The detection of Entamoeba histolytica and Toxoplasma gondii in wastewater[END_REF]. It would be necessary to develop eLAMP for other Bulinus or Biomphalaria species responsible for the transmission of Schistosoma. eLAMP could also be developed for other SBDs that have the same diagnostic pressures as schistosomiasis Bulinus truncatus snails are present in all sites except the water intake site in the Cavu River and university site in the Perpignan Channel. Three volumes of water (1, 3 and 5 l) from both the shore and the streambeds of the rivers were collected in all Corsican sites. Each line represents the amplification result from each site using LAMP, qPCR or ddPCR amplification method. qPCR and ddPCR results come from Mulero et al. [START_REF] Mulero | Environmental DNA for detecting Bulinus truncatus: a new environmental surveillance tool for schistosomiasis emergence risk assessment[END_REF]. A green membrane means that at least one quarter of the filtration membrane is positive for Bulinus truncatus presence and red membrane means that no quarter of the filtration membrane is positive. Empty cells mean that no filtration was done under these conditions

Fig. 1 Fig. 2

 12 Fig. 1 Specificity and LOD (limit of detection) of the Bulinus truncatus eLAMP (Bt-eLAMP) assay. Results obtained with agarose gel electrophoresis after 45 min of reaction. a Specificity; lanes 1-5, Bulinus truncatus DNA templates from five different localities (Diama, Lampsar, Mbane, Corsica and Saneinte); lane 6, Bulinus globosus; lane 7, Bulinus senegalensis; lane 8, Bulinus forskalli; lane 9, Bulinus umbilicatus; lane 10, Biomphalaria glabrata; lane 11, Bithynia tentaculata; lane 12, Ancylus fluviatilis; lane 13, Gyraulus laevis; lane 14, Physa acuta; lane 15, Potamopyrgus antipodarum; lane 16, Pisidium casertanum; lane C + , positive control (Bulinus truncatus DNA; 1 ng); lane C-, negative control (water as template). b LOD; lane 1-11, Bulinus truncatus DNA tenfold serial dilutions (10 ng/μL to 1 ag/μl); lane C-, negative control (water as template)

Fig. 3

 3 Fig. 3 Real-time loop-mediated isothermal amplification of Bulinus truncatus environmental DNA in Diama (a) and Lampsar (b) sites, Senegal. Orange, yellow and green lines are tested samples. Turquoise and blue lines are positive controls (1 ng of B. truncatus extracted DNA). Red line is field negative sample (1500 ml filtration of mineral water). Violet and pink lines are laboratory negative samples (ultrapure water).

Table 1 )

 1 , including four specimens of the same genus but different species (i.e. B. forskalii, B. globosus, B. umbilicatus and B. senegalensis) and other phylogenetically related genera. Bulinus truncatus from five different localities (Table

Table 1

 1 Snail species used for assessing the specificity of the developed Bulinus truncatus LAMP primer sets

Table 2

 2 Primer sequences designed for Bulinus truncatus eLAMP assays

Table 3

 3 Cross table comparing LAMP and qPCR analyses ofBulinus truncatus eDNA with kappa agreement

Acknowledgements

Special thanks to Alassane Ndiaye who helped collect the environmental DNA samples in the field. We thank Celia Robin who worked on the project during her internship and designed the additional LOOP primers.

Funding

This study was funded by the French Agency for Food, Environmental and Occupational Health & Safety (PNRES 2019/1/059 Molrisk) and the Occitanie Region (Schistodiag program). This study was carried out with the support of LabEx CeMEB, an ANR "Investissements d'avenir" program (ANR-10-LABX-04-01), and within the framework of the "Laboratoires d'Excellences (LABEX)" TULIP (ANR-10LABX-41).

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Declarations

Supplementary Information

The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s13071-023-05705-4.

Additional file 1.

Alignment of the Bt-eLAMP primers to the ITS2 marker (accession number: MG757890). Each arrow represents a primer. The green shades represent the direction of the primer hybridization (dark green: forward; light green: backward).

Author contributions JP, JB, OR and MB conceived the original idea and designed the study. MB performed the laboratory development of the Bt-eLAMP assay. JB, BS and MB carried out the field sampling. JB and BS provided malacological expertise. SM provided the eDNA bank. JB, JP and MB analysed and interpreted the data. JB and MB drafted the manuscript. BS, JB, SM, OR, JP and MB revised the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests