Charlie Franck

Alfred Compaoré

Hamidou Ilboudo

Jacques Kaboré

Justin Windingoudi Kaboré

Oumou Camara

Mohamed Bamba

Hassane Sakande

Minayégninrin Koné

Mamadou Camara

Dramane Kaba

Jacques Kaboré

Justin Windingoudi Kaboré

Adrien Marie

Gaston Belem

Stijn Deborggraeve

Philippe Büscher

Bruno Bucheton

Veerle Lejon

Vincent Jamonneau
email: vincent.jamonneau@ird.fr

Analytical sensitivity of loopamp and quantitative real-time PCR on dried blood spots and their potential role in monitoring human African trypanosomiasis elimination

Keywords: Trypanosoma brucei gambiense, diagnosis, dried blood spots, Loopamp, quantitative realtime PCR, sensitivity, feasibility

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Human African trypanosomiasis (HAT) or sleeping sickness is a parasitic disease caused by two subspecies of trypanosomes transmitted by tsetse flies [START_REF] Buscher | Human African trypanosomiasis[END_REF].

Trypanosoma brucei gambiense (Tbg) is responsible for a chronic disease (gambiense-HAT) in Central and West Africa and represents 98% of all reported HAT cases. The remaining 2% of HAT cases are due to Tb rhodesiense (Tbr) that causes an acute infection in East Africa [START_REF] Franco | Monitoring the elimination of human African trypanosomiasis: Update to 2016[END_REF]. Thanks to control efforts conducted in the last two decades (case detection, treatment and vector control), the number of reported cases has gradually decreased to fewer than 1000 cases in 2018 (https://www.who.int/trypanosomiasis_african/en/). HAT elimination is monitored through two primary global indicators: the annual number of reported cases and the area at risk reporting ≥ 1 case/10,000 people/year (calculated over a 5-year period) [START_REF] Franco | Monitoring the elimination of human African trypanosomiasis: Update to 2016[END_REF]. The objective set by World Health Organization (WHO) to reach the elimination of HAT as a public health problem by 2020 (WHO, 2012), with fewer than 2,000 reported cases annually and a reduction of 90% of the area at risk as compared to the 2000-2004 WHO baseline, is being achieved [START_REF] Franco | Monitoring the elimination of human African trypanosomiasis at continental and country level: Update to 2018[END_REF]. The indicator at country level for gambiense-HAT is the number of cases/10,000 people/year, in each health district of the country averaged over the previous 5-year period.

For formal validation by WHO of the elimination of HAT as a public health problem in a country, this indicator should be < 1, on condition that sufficient control and surveillance activities have been undertaken. This target has probably been met in some countries [START_REF] Franco | Monitoring the elimination of human African trypanosomiasis at continental and country level: Update to 2018[END_REF], which can request WHO to validate their status.

Adapted strategies and tools should be implemented to at least maintain this status, i.e. avoid re-emergence, and to reach the next objective set by WHO for gambiense-HAT: the interruption of transmission by 2030 [START_REF] Franco | Monitoring the elimination of human African trypanosomiasis: Update to 2016[END_REF]. It will therefore be crucial to monitor for potential transmission. As specialized local HAT control capacities will decrease or disappear, the use of highly specialized methods, such as parasitological tests, which are adapted to field condition but time-consuming, is excluded. In the future, monitoring tests will increasingly be performed in regional reference centres. Test specimens should therefore be easily collectable and safely transportable, such as dried blood spots (DBS) [START_REF] Sarkar | Dried blood spot for Ebola testing in developing countries[END_REF], while tests should be reliable, cheap and allow analysis of large numbers of samples in a high-throughput format.

Remote molecular testing of DBS has been proposed to assess the prevalence of HAT in a population, and a variety of molecular diagnostic tests is available [START_REF] Buscher | How can molecular diagnostics contribute to the elimination of human African trypanosomiasis?[END_REF]. The only commercialized method is the Loopamp Trypanosoma brucei Detection Kit (Eiken Chemical, Tokyo, Japan) [START_REF] Matovu | Towards Point-of-Care Diagnostic and Staging Tools for Human African Trypanosomiaisis[END_REF][START_REF] Njiru | Loop-mediated isothermal amplification technology: towards point of care diagnostics[END_REF], based on the loop-mediated isothermal amplification technology (LAMP) [START_REF] Notomi | Loop-mediated isothermal amplification of DNA[END_REF], and adapted for HAT diagnosis by amplifying the random insertion mobile element (RIME) [START_REF] Njiru | Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense[END_REF]. The quantitative real-time PCR (qPCR) technology [START_REF] Heid | Real time quantitative PCR[END_REF] was also applied to HAT diagnosis [START_REF] Becker | Real-time PCR for detection of Trypanosoma brucei in human blood samples[END_REF][START_REF] Mumba | Prevalence of human African trypanosomiasis in the Democratic Republic of the Congo[END_REF] and primers targeting the 18S ribosomal RNA gene were designed (M18S qPCR) [START_REF] Benfodil | Comparison of serological and molecular tests for detection of Trypanosoma evansi in domestic animals from Ghardaia district, South Algeria[END_REF].

Both Loopamp and M18S qPCR methods are Trypanozoon-specific. A Tbg specific method would be preferable to exclude the detection of Tb brucei (Tbb) that can potentially transiently infects humans in HAT endemic areas where Tbg and Tbb are sympatric [START_REF] Garcia | Follow-up of Card Agglutination Trypanosomiasis Test (CATT) positive but apparently aparasitaemic individuals in Cote d'Ivoire: evidence for a complex and heterogeneous population[END_REF][START_REF] Koffi | Aparasitemic serological suspects in Trypanosoma brucei gambiense human African trypanosomiasis: a potential human reservoir of parasites?[END_REF]. This is possible through detection of the Tbg-specific glycoprotein gene (TgsGP) [START_REF] Berberof | A receptor-like flagellar pocket glycoprotein specific to Trypanosoma brucei gambiense[END_REF][START_REF] Radwanska | Novel primer sequences for polymerase chain reaction-based detection of Trypanosoma brucei gambiense[END_REF], for which a qPCR has been developed [START_REF] Wagemans | Real-time polymerase chain reaction for diagnosis of human African trypanosomiasis[END_REF].

We compared the analytical sensitivity of the Loopamp, M18S qPCR and TgsGP qPCR, applied on DBS in the context of an upcoming diagnostic clinical trial evaluating diagnostic performance and feasibility of different diagnostic methods and algorithms for post-elimination monitoring (DiTECT-HAT-WP3, ClinicalTrials.gov Identifier:

NCT04099628). until reaching approximately 7.5 x 10 7 trypanosomes per mL of blood, as determined using an Uriglass® disposable counting chamber. Mouse blood was collected by cardiac puncture using a heparinized 1 mL syringe and diluted into phosphate buffered saline glucose (PSG, 38 mM Na2HPO4.2H20, 2 mM NaH2PO4.H2O, 83 mM glucose, 29 mM NaCl, pH 8.0) to achieve a stock concentration of 1 x 10 7 parasites/ml, which was kept on ice. Ten-fold serial dilutions were prepared in PSG to get concentrations ranging from 1 x 10 6 to 10 trypanosomes per mL in a total volume of 1 mL.

Material and Methods

Parasites

B4_C191 and B4_F303

Preparation of dried blood spots

Sixteen mLs of blood were collected from a human volunteer in Vacutainer heparinized tubes. Volumes of 450 µLs of blood were aliquoted in 32 microtubes. For each strain, 50 µL of each trypanosome suspension (1x10 7 to 10 trypanosomes per mL) were added in each tube. This was performed in duplicate, to obtain for each trypanosome strain, two blood sample series (A and B) with concentrations ranging from 1x10 6 to 1 trypanosomes per mL. For each series, a negative control was prepared by mixing 50 µL PSG with 450 µL human blood.

For both M18S and TgsGP qPCR, 30 µL of each trypanosome suspension and sampling control were spotted on Whatman 4 filter paper. For Loopamp, the sampling procedure described by Matovu et al. ( 2016) was followed with some modifications (https://www.finddx.org/wp-content/uploads/2016/06/HAT-LAMP-SOP_13JUN16.pdf).

Briefly, 90 µL of blood was mixed with 10 µL of 5% sodium dodecyl sulphate (SDS, Sigma) and incubated for 5 minutes at ambient temperature followed by spotting 40 µL on Whatman 1001 filter paper. All blood spots were air dried for one hour in a dry environment out of direct sunlight, and stored separately at room temperature for a maximum of 48 hours in closed plastic bags containing silica gel until DNA extraction.

DNA extraction

With a perforator, three discs of 5 mm diameter were punched out of each Whatman 4

DBS and out of one blank filter paper used as negative extraction control. Between each sample, the perforator was decontaminated with 1% hypochlorite, ultra-pure water and absolute ethanol. Each set of three discs were processed using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) following the instructions provided by the manufacturer.

Extracted DNA eluted in 50 µL elution buffer was stored at -20°C until qPCR analysis. For the Whatman 1001 DBS and a blank filter paper, DNA was extracted from one 5 mm diameter disc by adding 100 µL molecular biology grade water, mixing and incubating at 90°C for 10 minutes followed by centrifugation at 18,000 g for 3 minutes. These solutions containing the DNA were used for Loopamp reactions.

Quantitative real-time PCR and Loopamp reactions

All primers and probes are detailed in Table 1. qPCR reactions were performed using the TaqMan Probe assays. For M18S, forward and reverse primers were described in For Loopamp we followed the instructions provided with the kit. Positive and negative controls provided in the kit were included for each run. Reaction tubes were incubated at 65°C for 40 min in the LAMP Genie III (OptiGene Ltd) in which eight tubes can be processed per run. A result was considered positive when fluorescence was observed in the reaction tubes during exposure to a blue LED illuminator with a 470 nm ultra-violet light and confirmed by the kinetics of the fluorescence appearance detection by the integrated analysis system in the LAMP Genie® III instrument.

Results

M18S qPCR and TgsGP qPCR results for the ten-fold serial dilutions of strains B4_C191 and B4_F303 in duplicate are given in Table 2. No Ct value was obtained for the human blood without trypanosomes (negative sample controls), the extraction controls (blank filter paper) and the amplification reaction controls (water). For both strains, the lower detection limit in the duplicate runs was 1,000 and 10,000 trypanosomes/mL for M18S qPCR and TgsGP qPCR respectively.

For Loopamp, negative results were also obtained for negative controls (reaction, extraction and sampling) and positive results were obtained with positive controls validating the tests. The lower detection limit was 100 trypanosomes/mL for the two strains (Table 2).

Figure 1 illustrates the results obtained for B4_C191 Series B with fluorescence observed with ultra-violet light (Figure 1A andB) and with the kinetics of the fluorescence appearance (Figure 1C andD).

Discussion

Monitoring HAT elimination and post-elimination will probably rely on regional reference centers to overcome (i) the inevitable decrease of HAT control capacities in low endemic to zero transmission areas and (ii) the increasing non-adherence to the active HAT control activities of the communities who will no longer consider the disease as a threat. This requires the use of high-throughput methods applied on large numbers of samples easily collectable by non-specialized teams and easily transportable to be tested remotely. ). In the present study, the detection limit using M18S qPCR was only 1,000 trypanosomes/mL, which is probably due to the use of DBS as a decrease in sensitivity has been observed earlier when using such specimens [START_REF] Mumba | Performance of parasitological and molecular techniques for the diagnosis and surveillance of gambiense sleeping sickness[END_REF]. The limited volume of blood spotted in the DBS (30 µL) might explain this decrease.

Considering the low number of trypanosomes in blood of HAT patients, this analytic sensitivity casts doubt about the diagnostic sensitivity to be expected when analyzing clinical samples.

In the present study, the best analytical sensitivity was observed for the Loopamp test with a detection limit of 100 trypanosomes/mL. This is in line with the high sensitivity previously observed using RIME-LAMP methods for HAT diagnosis using freshly prepared samples [START_REF] Grab | Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP)[END_REF][START_REF] Kuboki | Loop-mediated isothermal amplification for detection of African trypanosomes[END_REF][START_REF] Mitashi | Diagnostic accuracy of loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples[END_REF][START_REF] Mugasa | Comparison of nucleic acid sequence-based amplification and loop-mediated isothermal amplification for diagnosis of human African trypanosomiasis[END_REF][START_REF] Njiru | Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense[END_REF], attributed to the high copy number of the targeted RIME sequence (500 copies/haploid genome) [START_REF] Bhattacharya | Mobile genetic elements in protozoan parasites[END_REF]. This is also in line with results obtained when using DBS of trypanosome-spiked blood samples treated with SDS in a recent experimental study carried out to optimize the procedures for Loopamp sample preparation that gave detection limit of 10 to 100 trypanosomes/mL depending on the storage time of DBS [START_REF] Matovu | Optimisation of template preparation and laboratory evaluation of the Loopamp Trypanosoma brucei kit for detection of parasite DNA in blood[END_REF]. Despite the fact that we adapted the original protocol, by reducing the amount of blood from 450 µL in the original protocol to 90 µL, envisaging fingerpick collection, the analytical sensitivity we observed is still in accordance with the low parasitaemia observed in HAT patients. Thanks to its simplicity, rapidity and good sensitivity, Loopamp appears to be the most promising DNA detection tool for HAT diagnosis performed on DBS.

However, the performance of a diagnostic method for large scale monitoring, also depends on its capacity to test a large number of specimens. Loopamp allows only eight specimens to be tested per run on the LAMP Genie III including the positive and negative control. The test duration for one reaction was around 1 hour and 30 minutes in our hands, including extraction, mix preparation and amplification time. We estimated the number of days to test 1,000 samples, for a trained lab technician working 7 hours per day, between 35 to 40 days. However, the analysis time could be reduced by using for example the Eiken LF-160 LAMP incubator that can take 16 tubes in a single run, or by changing for a 96 samples format such as for the Eiken Malaria LAMP kit [START_REF] Perera | Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria[END_REF], or by testing in multiple machines in parallel. Another drawback of Loopamp for a high-throughput use is the sampling procedure: a blood lysis step with SDS is required before spotting the specimen on Whatman 1001 filter paper. Similarly, the time-consuming DNA extraction step considerably reduces the speed of qPCR to maximum 1,000 samples per 10 days. The applicability of qPCR for high-throughput testing is therefore also questionable in its present form. Including HAT diagnosis in high-throughput platforms could overcome this limitation.

In the present study, we investigated the analytical sensitivity of the assays, known to be a potential challenge with DBS. However, collection of DBS in field conditions should take into account measures to mitigate risk of sample-to-sample contamination. Neither did we assess specificity of the assays for Trypanozoon or Tbg as this was already performed for both qPCRs and RIME LAMP elsewhere [START_REF] Njiru | Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense[END_REF][START_REF] Wagemans | Real-time polymerase chain reaction for diagnosis of human African trypanosomiasis[END_REF]. The effect of DBS storage time was not assessed. A decrease of sensitivity in Loopamp was recently observed with increasing storage time of the DBS, probably due to a progressive DNA degradation [START_REF] Matovu | Optimisation of template preparation and laboratory evaluation of the Loopamp Trypanosoma brucei kit for detection of parasite DNA in blood[END_REF]. Within an elimination monitoring context, storage time will largely be influenced by exterior logistic limitations, like time needed for DBS collection and for dispatching them to the reference laboratory, and is not entirely intrinsic to the assay. It can indeed be expected that performance of the assays under real life conditions, for several reasons, will be inferior than under ideal laboratory conditions. DNA degradation as observed elsewhere [START_REF] Matovu | Optimisation of template preparation and laboratory evaluation of the Loopamp Trypanosoma brucei kit for detection of parasite DNA in blood[END_REF] may have a serious impact on the accuracy of prevalence estimations with molecular tools. Alternatives, such as blood collected on DNA stabilization buffer, may also be considered [START_REF] Mumba | Performance of parasitological and molecular techniques for the diagnosis and surveillance of gambiense sleeping sickness[END_REF].

Other DNA detection methods previously described and applicable on DBS were not selected. For example, we did not choose the highly sensitive TBR primers targeting the 177bp repeat satellite DNA previously used in PCR [START_REF] Moser | Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction[END_REF] and qPCR [START_REF] Becker | Real-time PCR for detection of Trypanosoma brucei in human blood samples[END_REF][START_REF] Mumba | Prevalence of human African trypanosomiasis in the Democratic Republic of the Congo[END_REF] because of doubts about their specificity and the risk of contamination, in particular when many specimens need to be collected, transported and analyzed. The Tbg-specific methods such as 5.8S rRNA-internal transcribed spacer 2 LAMP [START_REF] Thekisoe | Preliminary application and evaluation of loop-mediated isothermal amplification (LAMP) for detection of bovine theileriosis and trypanosomosis in Tanzania[END_REF] or the TgsGP LAMP [START_REF] Njiru | Rapid and sensitive detection of human African trypanosomiasis by loop-mediated isothermal amplification combined with a lateral-flow dipstick[END_REF] were suspected not to be sufficiently sensitive. Also, the nucleic acid sequence-based amplification technology (NASBA) which is highly sensitive for HAT diagnosis [START_REF] Mugasa | Comparison of nucleic acid sequence-based amplification and loop-mediated isothermal amplification for diagnosis of human African trypanosomiasis[END_REF][START_REF] Mugasa | Detection of Trypanosoma brucei parasites in blood samples using real-time nucleic acid sequence-based amplification[END_REF], was not selected because of the need for RNA purification. Our results show that the challenge and the necessity of developing a molecular tool adapted to monitor HAT elimination still remains. The 7SL-sRNA target recently described as highly sensitive, and applicable to DBS might be a potential candidate although it is not Tbg-specific neither [START_REF] Chiweshe | Parasite specific 7SL-derived small RNA is an effective target for diagnosis of active trypanosomiasis infection[END_REF].

Developing a DBS based molecular tool adapted for monitoring HAT elimination is a challenge that relies on the sensitivity/specificity balance and the feasibility to test large numbers of samples. The three methods chosen for this study do not meet these requirements.

The TgsGP qPCR is in theory the most specific method but has low sensitivity. This was not completely unexpected, but as it follows the same methodology as the M18S qPCR, it was added to this study. The M18S qPCR is not Tbg-specific and its sensitivity is still not sufficient considering the parasitaemia observed in HAT patients. The high-throughput potential of both qPCR methods was disappointing. Loopamp has a better sensitivity but is not Tbg-specific and in its present form is also not entirely suitable for high-throughput use.

In

Figure 1 .

 1 Figure 1. Loopamp results for B4_C191 ten-fold serial dilutions (Series B) and controls Fig 1A and 1B illustrate the results observed with ultra-violet light. Fig 1C and 1D illustrate the kinetics of the fluorescence appearance. Results were obtained in two runs. Results obtained for 10 6 to 10 trypanosomes/mL (tryp/mL) are given in Fig 1A and Fig 1C and results obtained for 1 tryp/mL, human blood (sampling control) and extraction control are given in Fig B and Fig D. Positive and negative reaction controls were added for each run.

  in 25 μl final volume containing 5 μl of extracted DNA and 20 μl mix (1x FastMix II, 0.2µM each primer and TaqMan probe, 6.75 µL PCR grade water). The amplification conditions were as follows: polymerase activation at 95°C for 5 min, 45 cycles at 94°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. PCR assays were performed using the AriaMix

	Real-Time PCR system (Agilent Technologies) and the cycle threshold (Ct) values were
	automatically obtained. A result was considered positive if Ct < 40. A positive control (Tbg
	DNA) and negative control (water) were included in each reaction.

[START_REF] Deborggraeve | Diagnostic accuracy of PCR in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study[END_REF]

and the TaqMan probe was described in

[START_REF] Benfodil | Comparison of serological and molecular tests for detection of Trypanosoma evansi in domestic animals from Ghardaia district, South Algeria[END_REF]

. For TgsGP, primers and probe were described in

[START_REF] Wagemans | Real-time polymerase chain reaction for diagnosis of human African trypanosomiasis[END_REF]

. Amplifications were carried out

  The qPCR and LAMP technologies that previously showed high sensitivities in experimental and field studies (Buscher and Deborggraeve, 2015; Matovu et al., 2012; Mugasa et al., 2012) seemed promising tools for this purpose. In this study, we evaluated the analytical sensitivity of the

	Trypanozoon-specific M18S qPCR and Loopamp and the Tbg-specific TgsGP qPCR applied
	on DBS.
	In gambiense-HAT, blood parasitaemia is fluctuating and may be below the detection
	limit of 10 to 100 trypanosomes/mL of the most sensitive field-adapted parasitological test,
	the mini Anion Exchange Centrifugation Technique (mAECT) (Buscher et al., 2009; Camara
	et al., 2010). With a detection limit of 10,000 trypanosomes/mL, the TgsGP qPCR on DBS

seems not sufficiently sensitive for screening for HAT. The limited sensitivity of PCR targeting the Tbg-specific single-copy gene TgsGP was previously observed in experimental and field studies

[START_REF] Matovu | Phase II evaluation of sensitivity and specificity of PCR and NASBA followed by oligochromatography for diagnosis of human African trypanosomiasis in clinical samples from D.R. Congo and Uganda[END_REF][START_REF] Morrison | Use of multiple displacement amplification to increase the detection and genotyping of Trypanosoma species samples immobilized on FTA filters[END_REF]

and we show that the qPCR technology is not able to improve the detection limit. Our study confirms the better sensitivity of the PCR targeting the Trypanozoon specific multi-copy 18S ribosomal gene

[START_REF] Deborggraeve | Diagnostic accuracy of PCR in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study[END_REF][START_REF] Mumba | Performance of parasitological and molecular techniques for the diagnosis and surveillance of gambiense sleeping sickness[END_REF]

, which in a previous study, had an analytic sensitivity of 100 trypanosomes/mL blood

[START_REF] Mitashi | Diagnostic accuracy of loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples[END_REF]

Table 1 . Primers and probes used in M18S qPCR, TgsGP qPCR and Loopamp Method Primers/probe Gene Target group Sequences (5'-3')

 1 the past, several serological methods for screening for HAT through DBS testing have been proposed. Several versions of the Card Agglutination Test for Trypanosomiasis Tbg, but not necessarily active infection since antibody responses in general disappear very slowly (several years) after resolution of the infection[START_REF] Jamonneau | Revisiting the immune trypanolysis test to optimise epidemiological surveillance and control of sleeping sickness in West Africa[END_REF]. Combination of antibody detection tests followed by nucleic acid detection tests to confirm (or not) an active infection would take into account the limitations of both methods. We therefore recommend to investigate the diagnostic performance and feasibility of different algorithms, combining serological and molecular tests for elimination monitoring.

	(Magnus et al., 1978) adapted to DBS have been developed (Chappuis et al., 2002; Miezan et
	al., 1991). However, an important decrease in sensitivity was observed after DBS storage, and
	the methodology was not entirely suited for high-throughput use (Chappuis et al., 2005). For
	high-throughput screening for HAT on DBS, ELISA (Geerts et al., 2020; Hasker et al., 2010;

[START_REF] Lejon | A semi-quantitative ELISA for detection of Trypanosoma brucei gambiense specific antibodies in serum and cerebrospinal fluid of sleeping sickness patients[END_REF]

, and immune trypanolysis (Mumba et al., 2011; Van Meirvenne et al., 1995) seem more satisfactory. However, serological tests detect trypanosome specific antibodies, which indicate potential contact with

Acknowledgments

This study was conducted in the framework of the DiTECT-HAT project, which is part of the EDCTP2 programme supported by the European Union (grant number DRIA-2014-306-DiTECT-HAT). We acknowledge all the technicians from the HAT teams of CIRDES, Bobo-Dioulasso, Burkina-Faso.

Declaration of competing interest

The authors declare that they have no conflicts of interests.

Monitoring HAT elimination requires:

Easy collection and safe transport of large numbers of specimens in the field Testing with reliable, cheap and high throughput methods in regional reference centre


Samuel Frey

Claire Blanchard

Cécile Caillard

Delphine Drui

Antoine Hamy

Christophe Trésallet

Éric Mirallié
email: eric.mirallie@chu-nantes.fr

Éric Pr

Mirallié

Thyroid surgery in obese patients: a review of the literature

Keywords: obesity, bariatric surgery, thyroidectomy, morbidity, and opotherapy

Key points • Obesity is associated with an increased prevalence of nodular and neoplastic thyroid diseases. • The specific morbidity of thyroidectomy does not appear to be influenced by obesity. Post-operative complications are mainly urinary tract-related and wound complications, especially infectious. • Patients who have undergone bariatric surgery are at greater risk of severe hypocalcemia after total thyroidectomy: special attention must be paid to pre-operative vitamin D deficiencies, and peri-operative vitamin D and calcium supplementation must be systematically prescribed. • Conversely, an obese patient with persistent hypocalcemia following thyroidectomy should not undergo malabsorptive bariatric surgery. • Levothyroxine (T4) doses should be adjusted in obese patients and the starting dose should be based, not on the actual observed weight, but on the ideal weight. • The weight loss induced by bariatric surgery is associated with a reduction in T4 requirements. To treat hypothyroidism in patients postgastric bypass, a liquid form of T4 can be administered. • For patients undergoing radioactive iodine therapy, the dose of I131 should not be modified; bariatric surgery does not modify the absorption of iodine.

Thyroid surgery in obese patients: a review of the literature

Summary

The incidence of obesity in the general population has tripled in the past four decades. The number of bariatric surgeries performed annually has quadrupled in the past ten years due to this obesity epidemic. Concomitantly, the number of patients who are obese or who have undergone bariatric surgery and have become candidates for thyroid surgery has also increased considerably. Among patients undergoing thyroidectomy in the French population, the proportion of obese patients currently represents 10 to 20%. In addition, the frequency of benign and malignant thyroid pathologies seems to be increased by obesity, with a Hazard Ratio of around 1.8 for thyroid cancers in obese women. While obesity does not seem to influence the specific post-operative morbidity of thyroidectomy, a history of malabsorptive bariatric surgery should encourage the greatest caution in patients who need to undergo thyroid surgery since it is associated with a significant risk of severe hypocalcemia (> 60% in some studies) requiring intravenous calcium supplementation in about 20% of cases.

In the latter situation, peri-operative vitamin D-calcium supplementation is essential. In addition, the replacement dose of Levothyroxine (T4) intended for replacement must also be adapted in obese patients, calculated not from the actual weight but from the ideal weight. The objective of this review is to discuss the influence of obesity on the evolution of nodular and neoplastic thyroid pathologies, on the morbidity of thyroid surgery as well as on post-operative drug treatments. The impact of bariatric surgery on these different aspects is also discussed.

Introduction

The prevalence of obesity has tripled in the general population between 1975 and 2016 [START_REF] Angelantonio | Body-mass index and all-cause mortality: individualparticipant-data meta-analysis of 239 prospective studies in four continents[END_REF] and has become a major public health problem. If this trend continues, the number of obese candidates for thyroid surgery is likely to grow in similar proportion. Obese patients today represent 10 to 20% of thyroidectomized patients in the French cohorts [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF][START_REF] Blanchard | Impact of body mass index on post-thyroidectomy morbidity[END_REF].

The number of bariatric surgeries has quadrupled in the past ten years [START_REF] Debs | Trends of bariatric surgery in France during the last 10 years: analysis of 267,466 procedures from 2005-2014[END_REF] and it is logical to assume that obese patients or those who have undergone bariatric surgery will also be more numerous among patients requiring thyroid surgery.

Changes in stomach acidity in restrictive surgery such as sleeve gastrectomy (SG), combined with absorptive changes in malabsorptive surgeries like gastric bypass (GBP), can affect the bioavailability of many drugs [START_REF] Padwal | A systematic review of drug absorption following bariatric surgery and its theoretical implications[END_REF]. This could have an impact on the necessary dose of T4 for replacement therapy after total thyroidectomy, as well as on vitamin D and calcium supplementation, and even on the absorption of I 131 for radiotherapy after thyroid cancer.

The objective of this review is to study the influence of obesity on nodular and neoplastic thyroid pathologies, on the morbidity of thyroid surgery, and on necessary hormonal replacement or adjuvant treatments (iodine 131 ). The impact of bariatric surgery on these different aspects? is also discussed.

Does obesity influence the prevalence of thyroid conditions?

o Obesity and the prevalence of thyroid nodules Thyroid morphology and function are significantly influenced by obesity. The thyroid volume is greater in women with a body mass index (BMI) > 40 kg/m² compared to a control population [START_REF] Cappelli | Morbid obesity in women is associated to a lower prevalence of thyroid nodules[END_REF]. This volume is proportional to lean body mass, which increases along with fatty mass in the obese patient. Fatty mass is defined as the weight represented by fat in the body, measurable by techniques such as impedancemetry or biphotonic X-ray absorptiometry [START_REF] Pietiläinen | Agreement of bioelectrical impedance with dual-energy X-ray absorptiometry and MRI to estimate changes in body fat, skeletal muscle and visceral fat during a 12-month weight loss intervention[END_REF], while lean mass is calculated by subtracting fatty mass from the total body mass.

Regarding thyroid nodules, a 2019 case-control study of 67 severely obese patients paired with 67 control patients highlighted an increase in the prevalence of nodules, often multiple, in patients with a BMI > 35 kg/m² [START_REF] Siqueira | Thyroid nodules in severely obese patients: frequency and risk of malignancy on ultrasonography[END_REF]. In this study, 65% of the nodules in obese patients were purely cystic on ultrasound, with no malignancy. In contrast, in another study involving 108 obese women with a BMI >30 kg/m², non-cystic nodules were less common in obese patients, but were larger (0.9 +/-1.2 ml vs. 0.4 ± 0.5 ml, p <0.005) [START_REF] Cappelli | Morbid obesity in women is associated to a lower prevalence of thyroid nodules[END_REF]. This increase in nodular dystrophy and in the overall volume of goiter may be linked to obesityrelated insulin resistance [START_REF] Harikrishna | The impact of obesity and insulin resistance on thyroid cancer: A systematic review[END_REF].

o Obesity and prevalence of thyroid cancer

A link has been well established between obesity and many types of cancer (colon, breast, endometrium, kidney, esophagus, gastric cardia) [START_REF] Bianchini | Overweight, obesity, and cancer risk[END_REF] and has led some authors to study the incidence of thyroid cancer in obese people.

Compared to a control population, the echostructure of thyroid nodules in obese patients does not seem to be more frequently suspicious [START_REF] Cappelli | Morbid obesity in women is associated to a lower prevalence of thyroid nodules[END_REF]. Over the past decade, modern imagery has led to a considerable increase in the fortuitous detection of thyroid carcinomas (mainly microcarcinomas), but this does not explain the more frequent detection of larger or more advanced cancers, suggesting the influence of factors other than the more widespread use of imaging.

Studies in women using a BMI threshold of > 30 kg/m² to define obesity have shown a link between obesity and the risk of developing thyroid cancer (all histological types combined) (Hazard Ratio (HR) between 1.75 (95% CI: 1.03-2.94) and 1.86 (95% CI: 1.19-2.91)) [START_REF] Meinhold | Nonradiation risk factors for thyroid cancer in the us radiologic technologists study[END_REF][START_REF] Guignard | Alcohol drinking, tobacco smoking, and anthropometric characteristics as risk factors for thyroid cancer: a countrywide case-control study in New Caledonia[END_REF]. These results have been validated by several meta-analyses [START_REF] Maso | A pooled analysis of thyroid cancer studies. V. Anthropometric factors[END_REF][START_REF] Schmid | Adiposity and risk of thyroid cancer: a systematic review and meta-analysis: Adiposity and thyroid cancer[END_REF]. On the other hand, despite this increased prevalence, it has not been shown that thyroid cancers are associated with more severe histoprognostic characteristics or a higher risk of recurrence in obese people [START_REF] Paes | The relationship between body mass index and thyroid cancer pathology features and outcomes: a clinicopathological cohort study[END_REF]. This could be due to the fact that BMI does not take into account the fat distribution profile [START_REF] Paes | The relationship between body mass index and thyroid cancer pathology features and outcomes: a clinicopathological cohort study[END_REF]. Other studies using markers that take the obesity profile into account, such as visceral obesity [START_REF] Tulinius | Risk factors for malignant diseases: a cohort study on a population of 22,946 Icelanders[END_REF] or waist circumference [START_REF] Kitahara | Body fat distribution, weight change during adulthood, and thyroid risk in the NIH-AARP Diet and Health Study[END_REF],

have highlighted this increased risk of cancer and have underlined the link between visceral obesity and thyroid cancer.

In males, these results are more controversial because this correlation is inconsistent in various studies [START_REF] Oh | Effects of excess weight on cancer incidences depending on cancer sites and histologic findings among men: korea national health insurance corporation study[END_REF][START_REF] Engeland | Body size and thyroid cancer in two million Norwegian men and women[END_REF]. This may be related to the fact that men are less subject to thyroid cancer and therefore represent lower numbers in the studies.

Papillary cancer accounts for 80% of thyroid cancer cases. The results of previous studies that include all types of thyroid cancer combined, mainly reflect the relationship between obesity and this differentiated sub-type of thyroid cancer. When considering papillary cancers exclusively, a Korean study of 1,551 patients showed a four-fold increase in the incidence of papillary macrocancers (size > 1cm) when the BMI increases by 0.3 kg/m² per year (Odds ratio 4.00 [95% CI: 2.91-5.4]) [START_REF] Hwang | Annual average changes in adult obesity as a risk factor for papillary thyroid cancer: a large-scale case-control study[END_REF]. The severity of papillary cancer appears to be influenced by high BMI. Three retrospective studies in 2012, 2013 and 2014 involving 443, 2,057 and 1,216 patients, respectively, found that subjects with a BMI > 30 kg/m² had a greater risk of micro-invasion [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF][START_REF] Kim | Associations between body mass index and clinico-pathological characteristics of papillary thyroid cancer[END_REF] and were more often diagnosed at advanced stages (TNM 3 and 4) [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF][START_REF] Kim | Associations between body mass index and clinico-pathological characteristics of papillary thyroid cancer[END_REF][START_REF] Harari | Risk of Advanced Papillary Thyroid Cancer in Obese Patients[END_REF]. Patients with a high BMI also had a higher rate of persistent or recurrent cancer after surgery, but only for papillary macro-carcinomas (18.7% with BMI > 30 kg/m² vs. < 10% with BMI < 30 kg/m²) [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF].

Other histological types of thyroid cancer have been less studied. A 2015 metaanalysis of retrospective studies of papillary, follicular, medullary and anaplastic cancers reported a relative risk of 1.26 (95% CI: 1.15-1.39), 1.29 (95% CI: 1.08-1, 53), 0.5 (95% CI: 0.27-0.91) and 1.93 (95% CI: 1.23-3.03), respectively [START_REF] Wang | Obesity and risk of thyroid cancer: evidence from a metaanalysis of 21 observational studies[END_REF].

This meta-analysis seems to indicate that obesity is associated with a decreased risk of medullary cancer, whose histological origin and pathophysiology differ fundamentally from that of differentiated thyroid cancers [START_REF] Wang | Obesity and risk of thyroid cancer: evidence from a metaanalysis of 21 observational studies[END_REF]. The mechanisms involved in this "protective effect" are still unknown and deserve further study.

o Pathophysiology of thyroid cancer in the obese patient

Little is known about the pathophysiological mechanisms that might explain the association between obesity and thyroid cancer. They have mainly been discussed for papillary cancers. An elevated level of thyroid stimulating hormone (TSH) is known to promote carcinogenesis of differentiated thyroid tumors. However, up to 20% of clinically obese euthyroid patients have an elevated TSH level without modifying thyroid hormones [START_REF] Gajda | Diagnosis and treatment of thyroid disorders in obese patients -what do we know?[END_REF]. Elevated TSH (socalled hyperthyrotropinemia) is mainly caused by dysfunction of thyroid hormone feedback suppression of the pituitary secretion of TSH [START_REF] García-Solís | Thyroid hormones and obesity: a known but poorly understood relationship[END_REF], considered as an adaptive mechanism aimed at increasing energy expenditure and consequently preventing weight gain. In fact, in experimental studies, obese patients have a decrease in the expression of thyroid hormone receptors in the central nervous system and in peripheral tissues [START_REF] Fontenelle | Thyroid function in human obesity: underlying mechanisms[END_REF]. Leptin, an enteral hormone whose secretion is increased in the obese patient because it is proportional to the quantity of adipose tissue, also plays a central role in the elevation of TSH by directly stimulating the secretion of thyrotropin releasing hormone (TRH) in the hypothalamus [START_REF] Gajda | Diagnosis and treatment of thyroid disorders in obese patients -what do we know?[END_REF]. The increase in leptin concentration is also directly implicated in thyroid carcinogenesis [START_REF] Schmid | Adiposity and risk of thyroid cancer: a systematic review and meta-analysis: Adiposity and thyroid cancer[END_REF].

In addition, some authors have shown that insulin resistance, frequently associated with obesity, is implicated in the development of thyroid cancer by promoting the activation of the IGF1 receptor (Insulin Growth Factor 1) [START_REF] Harikrishna | The impact of obesity and insulin resistance on thyroid cancer: A systematic review[END_REF].

Finally, the difference in results observed between men and women indicates a possible role of gonadal hormonal levels. In this sense, the risk of cancer seems to be increased in obese menopausal women, due to the increased risk of visceral obesity after the onset of menopause [START_REF] Kitahara | Body fat distribution, weight change during adulthood, and thyroid risk in the NIH-AARP Diet and Health Study[END_REF][START_REF] Hwang | Annual average changes in adult obesity as a risk factor for papillary thyroid cancer: a large-scale case-control study[END_REF].

o Bariatric surgery and thyroid nodules Since bariatric surgery improves the main mechanisms involved in thyroid carcinogenesis in obese patients as discussed above, in particular, by decreasing the concentrations of leptin and TSH [START_REF] Gadiraju | Levothyroxine dosing following bariatric surgery[END_REF], could bariatric surgery influence the development of thyroid pathologies? To our knowledge, only one study has investigated whether GBP influences the risk of developing thyroid nodules [START_REF] Zhang | Effect of laparoscopic roux-en-y gastric bypass surgery on thyroid hormone levels in chinese patients, could it be a risk for thyroid nodules?[END_REF]. This study does not conclusively define the cancer risk after bariatric surgery, but suggests an increased risk of progression in size of preexisting nodules, particularly in patients whose plasma T3/T4 ratio is low after surgery. The impact of bariatric surgery on thyroid carcinogenesis therefore remains unknown to date.

What are the special features of thyroid surgery in the obese patient?

o Thyroidectomy in the obese patient While it is tempting to think that thyroid surgery in obese patients should be associated with more complications, few studies have evaluated this. The main results of these studies are summarized in Table 1. Only some of these reported morbidity related to overweight and obesity in thyroid surgery as the main objective the analysis [START_REF] Blanchard | Impact of body mass index on post-thyroidectomy morbidity[END_REF][START_REF] Harari | Risk of Advanced Papillary Thyroid Cancer in Obese Patients[END_REF][START_REF] Buerba | Thyroidectomy and parathyroidectomy in patients with high body mass index are safe overall: Analysis of 26,864 patients[END_REF][START_REF] Finel | Thyroidectomy in patients with a high BMI: a safe surgery?[END_REF][START_REF] Milone | Thyroidectomy in high body mass index patients: A single center experience[END_REF]. Their interpretation is limited by their retrospective nature [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF][START_REF] Harari | Risk of Advanced Papillary Thyroid Cancer in Obese Patients[END_REF][START_REF] Milone | Thyroidectomy in high body mass index patients: A single center experience[END_REF][START_REF] Specht | Characteristics of patients having thyroid surgery under regional anesthesia[END_REF][START_REF] Caulley | Risk factors for postoperative complications in total thyroidectomy: A retrospective, risk-adjusted analysis from the National Surgical Quality Improvement Program[END_REF]. Several of these studies lack power due to the infrequent nature of complications after thyroid surgery and the prior calculation of number of patients [START_REF] Blanchard | Impact of body mass index on post-thyroidectomy morbidity[END_REF][START_REF] Finel | Thyroidectomy in patients with a high BMI: a safe surgery?[END_REF][START_REF] Milone | Thyroidectomy in high body mass index patients: A single center experience[END_REF][START_REF] Specht | Characteristics of patients having thyroid surgery under regional anesthesia[END_REF][START_REF] Philippe | Feasibility criteria for total thyroidectomy in outpatient surgery[END_REF]. Two studies also included overweight patients (BMI 25-30 kg/m²) [START_REF] Finel | Thyroidectomy in patients with a high BMI: a safe surgery?[END_REF][START_REF] Milone | Thyroidectomy in high body mass index patients: A single center experience[END_REF]. Only one prospective American study of 18,825 thyroidectomized patients that included 7,425 obese patients [START_REF] Buerba | Thyroidectomy and parathyroidectomy in patients with high body mass index are safe overall: Analysis of 26,864 patients[END_REF] had a methodology that provided level 2 evidence, however the study did not specifically address the complications of thyroid surgery. The level of evidence from the other studies is 4.

In general, operative time for thyroidectomy increased with increasing BMI in the study by Buerba et al. [START_REF] Buerba | Thyroidectomy and parathyroidectomy in patients with high body mass index are safe overall: Analysis of 26,864 patients[END_REF], suggesting that the surgery is more difficult. Similar results were observed in a prospective French study and a retrospective American study [START_REF] Blanchard | Impact of body mass index on post-thyroidectomy morbidity[END_REF][START_REF] Specht | Characteristics of patients having thyroid surgery under regional anesthesia[END_REF]. Harari et al. reported a non-significant increase in operating time [START_REF] Harari | Risk of Advanced Papillary Thyroid Cancer in Obese Patients[END_REF]. The total time spent in the operating room is longer in the obese patient [29 and seems to be linked to a significant increase in the anesthetic induction time [START_REF] Harari | Risk of Advanced Papillary Thyroid Cancer in Obese Patients[END_REF].

The study by Buerba et al. showed that general post-operative complications were increased in the obese patient (Odds ratio 1.5 95% CI: 1.0-2.1); the main morbidity was related to incisional complications (Odds ratio 2.3 95% CI: 1.9-4.9) when the BMI was greater than 30 kg/m²), and to urinary tract infections (Odds ratio 2.2 95% CI: 1.1-4.4) when BMI was > 35 kg/m²) [START_REF] Buerba | Thyroidectomy and parathyroidectomy in patients with high body mass index are safe overall: Analysis of 26,864 patients[END_REF]. This increased risk was only present in obese patients older than 70 in a retrospective highpower study of 40,025 thyroidectomy patients, including 18,054 obese patients [START_REF] Caulley | Risk factors for postoperative complications in total thyroidectomy: A retrospective, risk-adjusted analysis from the National Surgical Quality Improvement Program[END_REF]. Results were not statistically significantly when the number of patients was lower [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF][START_REF] Harari | Risk of Advanced Papillary Thyroid Cancer in Obese Patients[END_REF].

Post-operative pain was not greater in the obese patient [START_REF] Blanchard | Impact of body mass index on post-thyroidectomy morbidity[END_REF].

Studies of the specific complications of thyroidectomy showed that hypocalcemia, whether transient or permanent, did not occur more frequently in obese patients, but the level of evidence was low [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF][START_REF] Blanchard | Impact of body mass index on post-thyroidectomy morbidity[END_REF][START_REF] Philippe | Feasibility criteria for total thyroidectomy in outpatient surgery[END_REF] show similar results in overweight patients [START_REF] Finel | Thyroidectomy in patients with a high BMI: a safe surgery?[END_REF][START_REF] Milone | Thyroidectomy in high body mass index patients: A single center experience[END_REF]. Only Trésallet et al. [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF] have shown an increased incidence of permanent (and not transient) recurrent nerve paralysis in their series (2.8% versus < 1% when BMI <30 kg/m²). This result, which could be explained by greater stretching of the recurrent nerve stretch due to increased operative difficulties, could also be linked to a higher preoperative prevalence of recurrent nerve paralysis due to more invasive cancers, as suggested by Harari et al. [START_REF] Harari | Risk of Advanced Papillary Thyroid Cancer in Obese Patients[END_REF]. The absence of pre-operative vocal cord examination in this last cohort as well as the lack of increased recurrent nerve paralysis in the study by Blanchard et al. [START_REF] Blanchard | Impact of body mass index on post-thyroidectomy morbidity[END_REF], study of thyroidectomy for benign pathologies, are arguments in favor of the second hypothesis.

Post-operative hematomas are not more frequent in obese [START_REF] Trésallet | The incidence of papillary thyroid carcinoma and outcomes in operative patients according to their body mass indices[END_REF][START_REF] Blanchard | Impact of body mass index on post-thyroidectomy morbidity[END_REF][START_REF] Philippe | Feasibility criteria for total thyroidectomy in outpatient surgery[END_REF] or overweight patients [START_REF] Finel | Thyroidectomy in patients with a high BMI: a safe surgery?[END_REF][START_REF] Milone | Thyroidectomy in high body mass index patients: A single center experience[END_REF].

It is important to take into account the fact that these studies focusing on the specific complications of thyroid surgery in the obese patient were carried out at expert centers, thus inducing a potential bias in patient selection of the included patients. The specific morbidity of thyroid surgery therefore deserves further study.

o Bariatric surgery and thyroidectomy Roux-en-Y GBP leads to decreased absorption of calcium and vitamin D, which takes place mainly in the duodenum and the proximal jejunum that are excluded by GBP. In combination with other factors such as lack of sun exposure, reduced caloric intake (and therefore vitamin D) after this surgery leads to an increase in the prevalence of vitamin D deficiency compared to that of the general population [START_REF] Spartalis | Post-thyroidectomy hypocalcemia in patients with history of bariatric operations: current evidence and management options[END_REF].

Strict attention must therefore be paid to the preservation of the parathyroid glands during total thyroidectomy in obese patients. Few studies have focused on the risk of hypocalcemia after thyroidectomy in obese people. The most recent systematic review of the literature [START_REF] Spartalis | Post-thyroidectomy hypocalcemia in patients with history of bariatric operations: current evidence and management options[END_REF] showed that this risk was only analyzed in 87 patients with a history of GBP, including numerous case reports.

The principal series are summarized in Table 2 although their level of evidence is low in view of the small number of patients included. Chereau et al. observed post-operative hypocalcemia in 60% of 15 post-GBP patients who underwent total thyroidectomy, compared to 15% in thyroidectomy patients with no history of bariatric surgery [START_REF] Chereau | Hypocalcemia after thyroidectomy in patients with a history of bariatric surgery[END_REF]. This hypocalcemia was permanent in 67% of the cases and required intravenous calcium supplementation in 27% of the patients. The studies of Dequanter et al. [START_REF] Dequanter | Recalcitrant hypocalcemia after total thyroidectomy and bariatric surgery[END_REF] and McKenzie et al. [START_REF] Mckenzie | Recalcitrant hypocalcemia after thyroidectomy in patients with previous Roux-en-Y gastric bypass[END_REF], which included 14 and 19 patients respectively, highlighted a higher risk of symptomatic hypocalcemia (38% and 21% respectively vs. 0% in the control patients). The duration of hospital stay was significantly longer in the obese, suggesting a difficult correction of hypocalcemia usually requiring intravenous supplementation (18% and 21% respectively). Conversely, Droeser et al. [START_REF] Droeser | Hypoparathyroidism after total thyroidectomy in patients with previous gastric bypass[END_REF] did not observe this increased risk of hypocalcemia in their cohort of 25 patients. The high rates of post-operative hypoparathyroidism in the control patients in this nationwide study (4.1% of the controls required intravenous treatment post-operatively and 4.7% were still receiving calcium supplementation six months after surgery) suggest that much surgery was performed in non-expert centers, making the comparison questionable with the interest group, whose patients were probably managed in expert centers.

The assessment of the risk of hypocalcemia after total thyroidectomy in post-GBP patients is therefore based on case reports or series with only small numbers of patients. There is no recommendation for the management of these patients. The authors believe that, given these risks, thyroid surgery should be performed before performing bariatric surgery. In this perspective, it would be appropriate for thyroid assessment to be part of the clinical work-up before bariatric surgery, with a TSH assay as well as a cervical ultrasound.

When thyroidectomy is necessary in a post-GBP patient, the implementation of vitamin D and calcium supplementation must be anticipated, if necessary intravenously. The recommendations proposed by several authors are summarized in Table 3. Special attention must be paid to vitamin D deficiency, which must be corrected in the pre-operative period, and to calcium supplementation, which should be systematically considered before thyroid surgery [START_REF] Mckenzie | Recalcitrant hypocalcemia after thyroidectomy in patients with previous Roux-en-Y gastric bypass[END_REF]. Some even recommend systematic intra-operative calcium supplementation [START_REF] Manco | Hypocalcemia complicating near-total thyroidectomy in patients with coexisting lipid malabsorption due to biliopancreatic diversion[END_REF][START_REF] Durr | Severe hypocalcemia complicating thyroid surgery after Roux-en-Y gastric bypass procedure[END_REF]. Some teams strongly recommend systematic postoperative treatment with calcium and calcitriol [START_REF] Manco | Hypocalcemia complicating near-total thyroidectomy in patients with coexisting lipid malabsorption due to biliopancreatic diversion[END_REF][START_REF] Goldenberg | Thyroidectomy in patients who have undergone gastric bypass surgery[END_REF]. Oral calcium supplementation must be taken with meals to promote absorption, and the use of antacids should be limited as much as possible to maintain gastric acidity conducive to calcium absorption [START_REF] Manco | Hypocalcemia complicating near-total thyroidectomy in patients with coexisting lipid malabsorption due to biliopancreatic diversion[END_REF]. Finally, when intravenous supplementation fails to correct hypocalcemia, it may be necessary to consider reversing the GBP [START_REF] Goldenberg | Thyroidectomy in patients who have undergone gastric bypass surgery[END_REF].

In post-GBP patients requiring thyroid surgery, the surgical strategy for preventing long-term hypocalcemia may include the possibility of sub-total thyroidectomy in order to guarantee preservation of the superior parathyroid glands. Conversely, the advisability of bariatric surgery in a patient with permanent post-thyroidectomy hypocalcemia must be questioned. In all likelihood, permanent post-thyroidectomy hypocalcemia should contraindicate malabsorptive bariatric surgery of the GBP type.

For patients who have undergone restrictive bariatric surgery like SG, the risk of hypocalcemia, although less studied, also seems to be increased. Chereau et al. noted hypocalcemia in 30% of these patients (n = 11). However, these results were not statistically significant compared to the control group (15%) [START_REF] Chereau | Hypocalcemia after thyroidectomy in patients with a history of bariatric surgery[END_REF]. The decrease in stomach acid associated with partial gastrectomy is thought to be associated with poor absorption of ionic calcium [START_REF] Goldenberg | Thyroidectomy in patients who have undergone gastric bypass surgery[END_REF].

After thyroidectomy, does thyroid hormone replacement therapy need to be modified in the obese patient? o Obesity and T4 medication

After total thyroidectomy, it is essential to assess the effectiveness of thyroid replacement therapy with Levothyroxine, a tetraiodothyroxine (T4) analog, by measuring the serum TSH level. The prescribed replacement dose of Levothyroxine is usually 1.6 µg/kg. However, several studies have shown that this dose/weight ratio should be reduced in the obese patient [START_REF] Glymph | Levothyroxine replacement in obese hypothyroid females after total thyroidectomy[END_REF][START_REF] Santini | Lean body mass is a major determinant of levothyroxine dosage in the treatment of thyroid diseases[END_REF]. Indeed, calculation of the necessary dosage of T4 should be based on the lean body mass [START_REF] Santini | Lean body mass is a major determinant of levothyroxine dosage in the treatment of thyroid diseases[END_REF] where T4 de-iodination into the active hormone triiodothyroxine (T3) takes place. Conversely, T4 requirement is not correlated with body fat. Basing dosage on total weight, which contains proportionally less lean mass in the obese subject, therefore leads to overestimation of the required T4 dose.

In line with this, the results of a retrospective low-powered study (32 patients) showed that at the usual replacement dose (1.6 µg/Kg), 47% of obese patients were hyperthyroid due to over-replacement (versus 20% of control subjects), and suggested that treatment should therefore be initiated at a lower dose in these patients (1.25 µg/kg) [START_REF] Glymph | Levothyroxine replacement in obese hypothyroid females after total thyroidectomy[END_REF]. In a retrospective study of 122 patients, Ojomo et al. [START_REF] Gkotsina | Improved levothyroxine pharmacokinetics after bariatric surgery[END_REF] proposed an adapted T4 replacement protocol that decreases as the BMI increases. Several studies nevertheless suggest that basing replacement dosage on ideal weight allows a more exact calculation [START_REF] Glymph | Levothyroxine replacement in obese hypothyroid females after total thyroidectomy[END_REF][START_REF] Santini | Lean body mass is a major determinant of levothyroxine dosage in the treatment of thyroid diseases[END_REF]. Using the formula [Ideal weight = 45.5 + 2.3 x ([height (cm]/2.54) -60)], Glymph et al.

proposed initiating T4 dosage in the obese patient at 2.3 µg/kg of ideal weight [START_REF] Glymph | Levothyroxine replacement in obese hypothyroid females after total thyroidectomy[END_REF].

o Bariatric surgery, replacement therapy and radioactive iodine therapy

The required replacement dose of T4 has been shown to be lower after restrictive surgery such as SG [START_REF] Gkotsina | Improved levothyroxine pharmacokinetics after bariatric surgery[END_REF], and was unchanged or reduced after malabsorptive Roux-en-Y GBP surgery [START_REF] Gadiraju | Levothyroxine dosing following bariatric surgery[END_REF]. The measurement of body composition shows that lean mass as well as fatty mass decrease after bariatric surgery, thereby decreasing the T4 requirements [START_REF] Fierabracci | Weight loss and variation of levothyroxine (L-T4) requirements in hypothyroid obese patients after bariatric surgery[END_REF].

Few studies have attempted to analyze the absorption of T4 after GBP, but plasma T4 concentration is not reduced after GBP, which indicates that absorption is not impaired [START_REF] Gkotsina | Improved levothyroxine pharmacokinetics after bariatric surgery[END_REF]. Indeed, intestinal absorption of T4 occurs mainly at the level of the jejunum and ileum, which are not affected by many types of bariatric surgery. The rare cases of hypothyroidism seen after bariatric surgery seem to be due mainly to decreased gastric acidity leading to poor dissolution of the drug. To avoid this, liquid formulations of Levothyroxine can be used [START_REF] Fallahi | TSH normalization in bariatric surgery patients after the switch from l-thyroxine in tablet to an oral liquid formulation[END_REF].

After resection of intermediate-and high-risk, differentiated thyroid cancers, radiotherapy with radioactive I 131 is routinely used to totalize thyroidectomy. Does the dosage of I 131 need to be adjusted after bariatric surgery? The study by Manousou et al. including 788 patients showed that urinary iodine excretion was not reduced after SG or GBP, indicating that there was no decrease in iodine absorption [START_REF] Manousou | Iodine status after bariatric surgery-a prospective 10-year report from the swedish obese subjects (SOS) study[END_REF]. There is therefore no scientific rationale indicating that the dosage of I 131 should be modified in obese patients who have undergone bariatric surgery.

Perspectives for the future

The feasibility of new thyroidectomy techniques, in particular the recent boom in trans-oral thyroidectomy, needs to be assessed. A recent study suggests that there is no obesity-related excess morbidity in robotic trans-oral thyroidectomy [START_REF] Tai | Obesity may not affect outcomes of transoral robotic thyroidectomy: subset analysis of 304 patients[END_REF]. The impact of obesity on the morbidity and efficacy of thermoablative techniques for benign thyroid nodules is worth studying. One team suggested that high BMI would have no impact on the effectiveness of radio frequency thermoablation [START_REF] Oddo | Quality of life in patients treated with radiofrequency ablation for thyroid nodules[END_REF], but this treatment needs to be evaluated on a larger scale in obese patients.

Conclusions

The increase in the prevalence of obesity in the general population has been accompanied by an increased frequency of nodular and neoplastic thyroid pathologies. The number of bariatric surgery patients has also increased.

There are no specific recommendations regarding the surgical management of thyroid disease in these patients. To minimize the risk of major post-operative hypocalcemia, thyroid surgery should precede bariatric surgery. If thyroidectomy is necessary after bariatric surgery, it should only be considered after normalization of the plasma vitamin D concentration. Even though obesity is associated with a longer operative time, it does not seem to be associated with an increase in specific thyroidectomy complications. Finally, the doses of replacement therapy in these patients should be adjusted based on ideal rather than actual weight. Finel et al [START_REF] Finel | Thyroidectomy in patients with a high BMI: a safe surgery?[END_REF] 2014 • Post-operative events including: hemorrhagic, respiratory, wound, and cerebrovascular complications, need for re-intubations, hypocalcemia, recurrent laryngeal nerve palsy;

Tables

• ** Post-operative events including: pulmonary, neurologic, cardiovascular, and infectious complications, rehospitalizations, operative site infections, wound complications the need for blood transfusion or reoperation, patient death;

• *** Undefined general post-operative morbidity

  . The prospective study by Finel et al. and the retrospective study by Milone et al. appeared to

  thyroidectomy; P : Partial thyroidectomy; NS: No statistically significant difference between groups for the criteria studied; RNP: Recurrent laryngeal nerve palsy; OD: Mean operative duration (from incision to closure); OR: Odds ratio ; CI 95%: 95% confidence interval

Table 1 . Studies reporting post-operative complications from thyroid surgery in obese and/or overweight patients Authors Year Design Population studied General complications Complications specific to thyroidectomy Others Trésallet et al. [2] 2014

 1

						• Hypocalcemia: NS
			Cohort study Retrospectiv Monocentric e	• Thyroidectomies for papillary cancer, (176 obese patients) • 1206 total patients (PT 4%)	-	• Post-operative NS • Permanent RNP: 1% vs bleeding:	-
						2,8% (BMI>30) p=0,01
	Blanchard et al. [3]	2019	Cohort study Prospective Multicentric	• TT for benign pathologies • 1310 patients (290 obese patients)	Post-operative pain: NS	• Transient and permanent RNP: NS • Hematoma: NS	Longer OD (107 ± 46.30 min in obese vs. 96 ± 40.08 min, p=0.0209)
	Harari et al [22]	2012	Cohort study Retrospectiv	• TT for papillary thyroid cancer	All complications combined*: NS (univariate analysis)	OD: NS

• Post-operative abscess: NS

• Transient RNP: NS

• Hypocalcemia: NS

Table 2 . Synthesis of published studies reporting the risk of hypocalcemia after total thyroidectomy in patients post-Roux-en-Y gastric bypass

 2

Goldenberg et al. [START_REF] Goldenberg | Thyroidectomy in patients who have undergone gastric bypass surgery[END_REF] Calcium citrate (1800-2500 mg/j) and vitamin D3 (400-800 mg/day) for 4-6 weeks before surgery Calcium citrate continued for 14 days, followed by calcium carbonate for another 7-14 days