Assessing improved surveillance of leprosy drug resistance in Madagascar: A pilot study in the Miandrivazo district

Assessing improved surveillance of leprosy drug resistance in Madagascar: A pilot study in the Miandrivazo district | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessing improved surveillance of leprosy drug resistance in Madagascar: A pilot study in the Miandrivazo district Luca MAHARAVO, Stéphanie Ramboarina, Sofie Marijke Braet, Paul Corstjens, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9335540/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background: To curb the emergence of antibiotic-resistant Mycobacterium leprae strains, the WHO recommends monitoring of antimicrobial resistance (AMR). In Madagascar, molecular surveillance of resistance to anti- M. leprae drugs, remains limited geographically and in terms of quantitative data. A clinical trial on leprosy prophylaxis (PEOPLE) conducted in the district of Miandrivazo provided an opportunity to explore the feasibility of an integrated approach for improving leprosy surveillance and resistance monitoring in Madagascar. This study aimed to i) determine the genetic diversity of the identified M. leprae strains, (ii) detect mutations associated with AMR, (iii) evaluate and compare the acceptance rate of low-invasive sampling methods with that of skin biopsies, and (iv) assess seropositivity for anti- M. leprae PGL-I IgM. Methods: Patients were identified through door-to-door screening and skin biopsies (SB), fingerstick blood (FSB) and nasal swabs (NS) were collected. SB were analyzed by RLEP qPCR for detection of M. leprae DNA, resistance testing using LiPA (Line Probe Assay), and resistance analysis and SNP and/or VNTR genotyping using Sanger sequencing and Deeplex Myc-Lep. NS and FSB samples were used to detect bacterial colonization by RLEP qPCR and detect past and/or present infection by measuring anti- M. leprae PGL-I IgM levels. Results: 80 leprosy patients were identified, including 41 multibacillary and 39 paucibacillary. Out of these, 57 accepted NS, 47 FSB, and 33 SB. High levels of anti-PGL-I IgM were detected in 6/47 FSB samples. RLEP qPCR was positive in 4/57 NS samples and 28/33 SB samples. Among SB positive, 12 yielded interpretable results when combining data from LiPA, Deeplex Myc-Lep, and Sanger sequencing. No drug resistance conferring mutations were detected. All strains belonged to the 1D-Malagasy genotype, and VNTR analysis for 7 samples with results available across 9 loci showed 7 unique types, with loci GAA21, GTA9, and AC8a were the most informative for strain differentiation. Conclusion: Despite the small sample size, this study shows that NS and FSB samples are well accepted in patients. No mutations conferring resistance were identified and the M. leprae strains belong to the 1D-Malagasy genotype. These findings support the feasibility of field-based molecular surveillance and provide a foundation for expanding AMR monitoring in Madagascar. Mycobacterium leprae Madagascar RLEP qPCR Drug resistance Genotype 1D-Malagasy anti-PGL-I serology Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Leprosy, caused by Mycobacterium leprae or M. lepromatosis , is a chronic granulomatous disease primarily affecting the skin, peripheral nerves, and mucous membranes. The introduction of multidrug therapy (MDT), combining dapsone, rifampicin, and clofazimine, has significantly reduced the global prevalence of leprosy. However, over the past ten years, the average annual number of new cases has remained stable, with 172,717 new cases and 4,488 relapses reported in 2024 across more than 188 countries, reflecting the persistence of transmission. Furthermore, the emergence of drug-resistant leprosy has been reported in several endemic countries, prompting the World Health Organization (WHO) to recommend vigilant monitoring of resistance to key anti-leprosy medications, particularly among multibacillary (MB) patients [ 1 ]. The 2009–2015 surveillance of 1932 cases of anti-leprosy drug resistance (1143 relapse and 789 new cases) in 19 countries reported that 8.0% of M. leprae strains were resistant to anti-leprosy drugs. Regarding rifampicin, resistance was observed in Brazil (≈ 9.1%), India (≈ 4.7%), Colombia (≈ 24.3%), and Indonesia (≈ 3.3%) [ 2 ]. In Madagascar, approximately 1,500 new cases are still detected annually, of whom 10% are children under 15 years old, indicating ongoing transmission [ 3 ]. In 2021, 1,340 patients received treatment, highlighting the persistent disease burden [ 4 ]. In 2015 the Centre d’Infectiologie Charles Mérieux (CICM), the national reference laboratory for leprosy, began molecular monitoring of anti-leprosy drug resistance, in collaboration with both the national leprosy program and international partners. This surveillance was initially focused in Antananarivo and some leprosy endemic regions, thereby reducing the geographical representation of the data gathered. Currently, only one case of drug resistance has been recorded, based on the screening of high-risk patients recommended by clinicians (approximately 5–10 cases per year), which may indicate insufficient testing coverage rather than an actual lack of resistant strains [ 5 ]. In addition, genotyping studies of M. leprae strains have identified the 1D-Malagasy genotype, defined by the A1015G SNP in the gene ml2446 encoding the lipoprotein Q (LprQ), as the hyperdominant strain type circulating in these settings [ 6 ]. Nevertheless, among the 30 samples analyzed in that study, only two originated from the Menabe region, which includes the Miandrivazo district, a hyperendemic area for leprosy in western Madagascar. From 2018–2023, Madagascar participated in the PEOPLE study (Post-Exposure Prophylaxis for Leprosy), a randomized clinical trial ( https://clinicaltrials.gov/ct2/show/NCT03662022 ) designed to evaluate the efficacy of different post-exposure prophylaxis (PEP) strategies against leprosy. In Madagascar, the Miandrivazo district was selected as the study site based on its high incidence, to assess the effectiveness of single dose of rifampicin (SDR) administered prophylactically to contacts of leprosy patients [ 7 , 8 ]. This study has allowed identification of both new and previously diagnosed leprosy cases [ 9 ], providing a unique opportunity to assess resistance to anti-leprosy drugs in this area, particularly rifampicin, the drug endorsed by WHO for post-exposure prophylaxis. Given that rifampicin is applied in tuberculosis (TB) and leprosy health care, both for treatment and prophylaxis, it is important to survey the presence of resistant strains to prevent their selection or spread [ 10 ]. Since M. leprae cannot be cultured in vitro and phenotyping based on culture in the mouse footpad model is lengthy and costly, molecular tests have been developed to directly detect, from clinical samples, mutations associated with antimicrobial resistance [ 11 , 12 ]. To select samples for resistance testing, the WHO recommends prioritizing MB patients with bacteriological index (BI) ≥ 2, as these samples are more likely to be PCR-positive [ 1 ]. A BI ≥ 2 corresponds approximately to the minimal threshold of 10⁴ bacilli per gram of tissue required for reliable detection of acid-fast bacilli (AFB) by conventional microscopy [ 13 ]. Consequently, this method exhibits limited sensitivity, and by definition negative in paucibacillary (PB) patients. In contrast, the RLEP qPCR assay stands out as a sensitive molecular tool capable of identifying M. leprae DNA, even in samples with low bacterial counts [ 14 – 17 ]. Cycle threshold (Ct) values from RLEP qPCR also enable precise quantification of M. leprae genome copy numbers in the DNA extracts. Because sufficient bacterial DNA is required for molecular drug-resistance analyses, RLEP qPCR facilitates the identification of samples eligible for detection of possible drug resistance-associated mutations. Molecular testing of resistance of M. leprae to rifampicin, dapsone and fluoroquinolones is based on known resistance mutations in regions of the rpoB , folP1 , and gyrA/gyrB genes, respectively. Available methods include the GenoType Leprae DR (Hain Lifescience/Bruker) line probe assay based on post-PCR reverse hybridization [ 18 ], targeted Sanger sequencing [ 19 ], and the recently developed targeted next-generation sequencing Deeplex-Myc-Lep assay (GenoScreen)[ 20 ]. In addition to resistance testing and identification of M. leprae (and M. lepromatosis ), this assay simultaneously provides high-resolution genotyping based on multiple canonical typing SNPs and variable number of tandem repeat (VNTR) loci. Deeplex Myc-Lep has successfully been used to conduct a first countrywide drug resistance survey in the frame of the PEOPLE study, in the Comoros[ 21 ]. In addition to drug resistance detection, the PEOPLE study provides, for the first time in Madagascar, an opportunity to compare acceptance rates of low-invasive sampling methods, including nasal swabs (NS) and finger stick blood (FSB), with skin biopsies (SB) among leprosy patients. RLEP qPCR performed on NS allows for the detection of bacterial colonization, while FSB enables the measurement of anti- M. leprae PGL-I (phenolic glycolipid I) IgM, a biomarker specific for past and present M. leprae infection. The anti-PGL-I UCP-LFA test, used to quantify anti- M. leprae PGL-I IgM from minimally invasive capillary blood samples (FSB), was applied to 1,815 contacts of leprosy patients in our cohort. Analyses revealed a marked reduction in the seroprevalence of anti-PGL-I IgM, decreasing from 27.4% to 11.6% between the first and third year following the prophylactic intervention [ 22 ]. In leprosy patients, it has also been widely demonstrated that IgM levels directed against the PGL-I of M. leprae are strongly correlated with bacterial load and generally decrease significantly after effective treatment [ 23 – 25 ]. This biological sub-study of the PEOPLE project aimed to (i) determine the genetic diversity of the identified M. leprae strains isolated from the patients using VNTR and SNP analyses after RLEP qPCR-based selection (ii) examine the presence of resistance-associated mutations in target gene regions using LiPA, targeted Sanger sequencing and Deeplex Myc-Lep (iii) evaluate and compare the acceptance rate of low-invasive sampling (NS and FSB) with that of SB and (iv) asses seropositivity for anti- M. leprae PGL-I IgM in leprosy patients from the Miandrivazo district in Madagascar. Methods Study population and clinical samples Clinical samples and associated patient data were obtained from the PEOPLE study conducted in the Miandrivazo district. Patient screening was performed through a door-to-door approach in the community. The study was implemented in three rural communes in the southern part of the district Ankotrofotsy, Manambina, and Ambatolahy selected for their high endemicity, with an annual prevalence estimated at 1.5 cases per 10,000 population in 2018. Leprosy diagnosis followed the criteria of the WHO: patients presenting 1–5 skin lesions were classified as PB, and those with more than 5 lesions as MB [ 26 ]. Sampling SB, NS, and FSB samples were collected from all consenting leprosy patients between 0 and 10 months after MDT initiation (Fig. 1 ). NS samples were collected using ESWAB® kits (COPAN Italia, Cat. 013708), in accordance with the manufacturer's recommendations. SB were performed using a 4 mm punch after local anesthesia and placed in a microtube containing 1 mL of Disolol. However, SB were not performed in patients with neural forms. For FSB, 20 µl of blood were collected following the detailed protocols described in a previous study [ 27 ]. Timely sampling of all clinically confirmed cases was impeded by seasonal flooding and COVID-19 restrictions in 2020–2021, limiting field activities to a few dry-season periods between 2019 and 2021. All samples were promptly stored in a cold box in the field (4–10°C) and subsequently kept at 4°C at Miandrivazo hospital until transportation by road in a refrigerated box (4–8°C) to the National Reference Laboratory for Leprosy at the CICM in Antananarivo for analysis. Clinical diagnosis was performed based on WHO classification. Upon obtaining patient consent, samples were collected using skin biopsy and/or nasal swab and/or fingerstick blood (FSB). RLEP qPCR was used to detect M. leprae in skin biopsies and nasal swabs. The UCP-LFA was used to quantify anti-PGL-I IgM antibodies in FSB samples. 26/28 M. leprae positive skin biopsy samples were tested for drug resistance and strain genotype (2 samples excluded due to insufficient volume). Among the 26 samples, 12 yielded interpretable results using at least one of 3 techniques. The 14 failed (undetermined) results indicate Ct > 27. Nasal swab DNA extraction DNA extraction was performed using thermal shock with 200 µL of the sample. The inactivation step was carried out at 99°C for 30 minutes in a thermomixer (Eppendorf, Cat. 5355), followed by the addition of 1 µL of a pre-optimized Sample Processing Control (SPC) ([Yakima Yellow®-BHQ-1® Probe] 200 RXN, Eurogentec, RT-SPCY-B02) diluted at 1:10. Then, a thermal shock cycle at 96°C for 2 minutes was applied, followed by cooling on ice for 2 minutes, repeated five times [ 28 ]. Finally, the sample was incubated at 96°C for 15 minutes. And after a brief centrifugation of one minute, the supernatant containing the extracted DNA was recovered and then stored at -20°C. Skin biopsy DNA extraction Biopsy was finely fragmented and transferred into a tube GentleMACS M containing 500 µL of phosphate-buffered saline (PBS, Sigma, Cat. P4417) and 1 µL of a pre-optimized SPC diluted at 1:10 (from the qPCR DNA extraction and inhibition control kit [Yakima Yellow®-BHQ-1® Probe] 200 RXN, Eurogentec, RT-SPCY-B02). Tissue homogenization was performed using the gentleMACS dissociator (MACS-Miltenyi Biotec) with the RNA_02 program, followed by centrifugation at 2000 rcf for 5 minutes [ 29 ]. Subsequently, 200 µL of the homogenate was subjected to chemical lysis and DNA purification using the QIAamp UCP-Pathogen DNA MiniKit, following the manufacturer’s protocol (QIAGEN, Cat. 50214). The purified DNA was eluted in 100 µL of TE buffer and stored at -20°C. RLEP qPCR assays All DNA extracts from biopsy samples and NS were subjected to quantitative PCR (qPCR) targeting the RLEP repeat sequence to detect and quantify M. leprae , as previously described, with minor modifications [ 14 ]. The qPCR reaction was performed in a final reaction volume of 20 µl, containing 1X SensiMix II No-Rox (Bioline, Cat. Bio-83005), 400 nM each of forward and reverse primer, 100 nM of the probe, 300 nM ROX (to improve signal stability), 2X EGT control mix (Eurogentec, Cat. RT-IPCY-B02), and 2 µL of purified extracted DNA. Amplification was performed in a Bio-Rad CFX96 thermocycler, starting with an initial denaturation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds and 60°C for 60 seconds, with a final step at 4°C for 1 minute. Each sample was analyzed in analytical triplicate following published protocols [ 14 ]. For each qPCR run, nuclease-free water was included as a contamination control, along with a serial dilution of the M. leprae reference strain NHDP (3 × 10⁶ to 30 RLEP copies) (BEI, Ref. No. 19350). Based on the Ct value, the slope of the regression line, and the Y-intercept, the Bio-Rad CFX96 software automatically calculated the RLEP copy number. Anti- M. leprae PGL-I IgM UCP-LFA . Serological analysis of FSB collected in the field was performed using lateral flow assay (LFA) strips with integrated luminescent upconverting reporter particles (UCP) to quantitatively detect anti- M. leprae PGL-I IgM, following previously described protocols [ 22 , 27 , 30 , 31 ]. The results are expressed as normalized values (Ratio (R)), equivalent to the ratio between the fluorescence signal measured at the Test line and at the Flow Control line on the UCP-LFA test strip. Line Probe Assay All M. leprae -positive samples (RLEP Ct < 36) were tested using the GenoType Leprae DR test (Hain Lifescience, IFU-320-06), following the manufacturer’s instructions. The target regions included the rifampicin resistance-determining region in rpoB from codon 432 to 458, the quinolone resistance-determining region in gyrA from codon 89 to 91, and the dapsone resistance-determining region in folP1 from codon 53 to 55 [ 18 ]. Sanger sequencing Sanger sequencing was used to confirm the Line Probe Assay results and to target the specific 1D-Malagasy genotype. We used the primers recommended by the WHO to detect mutations in the rpoB , gyrA , and folP1 genes [ 32 ]. For the identification of the specific 1D-Malagasy locus ( ml2446 ), associated with the A1015G mutation, we used the primers described by Avanzi et al [ 6 ]. Amplification of the target genes was performed following the protocol of Avanzi et al. [ 6 ], with minor modifications. The PCR reaction was carried out using TopTaq Master Mix Kit (Qiagen, Cat.200403), and the amplicons were sequenced by the Sanger platform at GenoScreen ( https://www.genoscreen.fr/fr/ ). Results were analyzed using MEGA v7.1 software, comparing the detected mutations with those known to confer antibiotic resistance, as described by the WHO for the three studied genes. Deeplex Myc-Lep For the Deeplex® Myc-Lep test, the 10 samples with a RLEP Ct < 30 were sent to the “Mycobacteriology Service at Bichat-Claude Bernard Hospital in France”, where laboratory analyses were conducted in strict accordance with the manufacturer’s recommendations, as previously described [ 20 ]. The Deeplex® Myc-Lep assay (GenoScreen, GNSRUOLEP01-20) relies on deep sequencing of a single 44-plex amplicon mix for the simultaneous identification of mycobacterial species (based on the hsp65 gene sequence), genotyping (based on SNPs in 18 gene regions and 11 VNTR markers), and prediction of M. leprae drug resistance. Resistance prediction is based on the detection of known resistance-associated mutations in seven genes: rpoB , ctpC , and ctpI (rifampicin); folP1 (dapsone); and gyrA , gyrB (fluoroquinolones) and nth [ 20 , 21 ]. VNTR profiles were analyzed with R, visualized with a heatmap, and grouped by hierarchical clustering using Euclidean distance and the UPGMA method. Results Cohort characteristics and sampling Overall, 80 patients presenting clinical signs of leprosy were included, comprising 41 MB and 39 PB cases (Table 1 ). Table 1 Demographic and clinical information of leprosy patients. Patient characteristics before treatment (n = 5) during treatment (n = 75) total (n = 80) gender male 3 52 55 female 2 23 25 age (years) 50 1 23 24 WHO classification MB 3 38 41 PB 2 37 39 samples SB 4 29 33 NS 3 54 57 FSB 1 46 47 n indicates the number of patients. MB: multibacillary; PB: paucibacillary.; SB: skin biopsies; NS: nasal swab; FSB: finger stick blood. The cohort was predominantly male 55/80 with a mean age of 41 years, including six children under 16 years. Among the newly diagnosed cases, 5/80 were not under treatment at the time they were sampled, while 75/80 were already receiving MDT for 3 to 10 months (Table 1 ). Regarding sample collection 62/80 patients agreed to provide at least one sample of SB, FSB, or NS (Fig. 1 ). 33/80 patients (41%) consented to SB, 57/80 (71%) to NS sampling, and 47/80 (59%) to FSB sampling (Fig. 2 B). Acceptance of all three samples simultaneously (SB, NS, and FSB) was 41% (Fig. 2 A). Moreover, among the 80 patients, 25 (31%) presented with pure neural leprosy (17 neuro-MB, 8 neuro-PB). Acceptance rates were 56% (14/25) for FSB, 84% (21/25) for NS, and 8% (2/25) for SB (Supplementary Table 5). The SB was performed only in two patients with pure neural leprosy without skin lesions, for whom nerve-level SB above the knee were conducted with patient consent, in accordance with national guidelines. RLEP qPCR for skin biopsies Among the 33 patients who consented to SB sampling, 10/13 PB patients and 18/20 MB patients tested positive by RLEP qPCR (Table 2 ), yielding an overall positivity rate of 85%. The median Ct values were 30.0 (IQR 24.0–32.1) for MB patients and 32.0 (IQR 30.9–33.7) for PB patients. SB RLEP qPCR results demonstrated that MB patients carried significantly higher concentrations of M. leprae DNA than PB patients (p = 0.03) (Fig. 3 A), consistent with the expected relationship between bacterial load and clinical form. Analysis of bacterial load over treatment showed that Ct values exhibited no significant temporal trend in PB patients (nptrend z = − 0.05, p = 0.96) or MB patients (nptrend z = 1.03, p = 0.30), although values for MB tended to increase with treatment duration (Supplementary Table 3). These results showed the high sensitivity of RLEP qPCR for detecting M. leprae DNA during and after MDT, confirming leprosy before treatment in one PB and three MB patients. However, a DNA-based PCR test cannot distinguish viable from non-viable bacilli, particularly during the first months of MDT when M. leprae is rapidly killed (< 3 months), yet DNA can linger for years [ 33 ]. Table 2 : Diagnostic test data for treated and untreated leprosy patients. Patients classified as multibacillary (MB) and paucibacillary (PB), and according to treatment status at the time of sampling (untreated vs treated for 3 to 10 months). N: total number of patients; n: number of patients tested per technique. Results are expressed as the number of positives and negatives for Mycobacterium leprae detection by RLEP qPCR skin biopsies (SB), nasal swabs (NS) and as seropositive/seronegative for anti-PGL-I IgM by finger stick blood (FSB). RLEP qPCR RLEP for nasal swabs RLEP qPCR was positive in 4 of the 57 patients who consented to NS sampling, all of whom had MB leprosy with Ct values of 27.78, 28.96, 33.35, and 31.93. These patients had highest skin bacterial loads with Ct values of 14.01, 14.27, 16.6, and 25.32 in the RLEP qPCR performed on the concurrent SB samples (Supplementary Table 3). These results suggest that the detection of M. leprae in NS is primarily observed in MB patients with a high bacterial load as previously described [ 25 ] . Anti-PGL-I IgM levels in FSB Among the 47 patients who consented to FSB sampling (Table 2 ), only one had not yet started MDT at the time of sampling and was seronegative for anti-PGL-I IgM. Table 3 Demographic, clinical, and diagnostic data of six patients highly seropositive for anti-PGL-I IgM. ID Months of MDT before sampling Sex Age (years) Clinical signs Ct SB RLEP qPCR SB Ct NS RLEP qPCR NS PGL-I IgM (R) 9013 6 M 32 dMB 16.6 pos 33.34 pos 3.59 9073 4 M 10 nMB NT NA ND neg 1.30 9048 4 M 10 dMB 27.33 pos ND ND 1.29 9024 6 M 38 dMB NA ND 0 neg 0.97 9028 5 M 20 dMB 29.69 Pos 0 neg 0.76 9030 6 M 42 nPB NA ND 0 neg 0.36 NA: not applicable (no samples available as patients did not give consent); d: dermatological; n: neural; MB: multibacillary; PB: paucibacillary; neg: negative; pos: positive; Ct: cycle threshold; ND: not determined; M: male; NS: nasal swab; SB: skin biopsy; R: ratio between the fluorescence signal measured at the test line and at the flow control line on the UCP-LFA test strip. The other 46 were undergoing MDT treatment at the time of collection (Supplementary Table 3), of whom six (1 PB; 5 MB) were seropositive for anti-PGL-I IgM (Fig. 3 B). Among these 6 seropositive patients, 4 were detected positive for M. leprae by RLEP qPCR on SB samples. The two remaining patients are a neuro-MB and neuro- PB, for whom no SB could be performed. They were 42 and 38 years old, respectively, both at the 6th month of MDT, with anti-PGL-I IgM levels (R value) of 0.97 and 0.36 (Table 3 ). The FSB sample from MB patient, aged 32 years, presented an high R-value of 3.59 and displayed Ct values of 16.6 for the SB. and 33.34 for the NS in RLEP qPCR (Table 3 ), which is in line with previous findings [ 29 ] which reported a correlation between molecular and serological test result. Drug resistance RLEP qPCR was used as a pre-screening test for further molecular antibiotic resistance analyses, depending on Ct values, which were performed using three complementary techniques: LiPA, Sanger sequencing, and Deeplex Myc-Lep. Among the 26 samples tested with all three methods, 6/26 (with a Ct < 27) yielded interpretable results across all techniques (Supplementary Table 4). Three samples provided interpretable results only with Deeplex Myc-Lep, while three others, analyzed by LiPA and Sanger sequencing but not submitted to Deeplex Myc-Lep due to a Ct above the recommended threshold (Ct < 27), also produced usable results. Performance decreased markedly above Ct of 27, particularly for LiPA and Sanger sequencing, likely due to a too low bacillary load. Of the 26 DNA samples extracted from SB, 12 yielded interpretable results using at least one of the employed techniques (Supplementary Table 4). No mutations (Deeplex Myc-Lep and Sanger sequencing) or no known mutations (LiPA) in the rpoB , folP1 , or gyrA genes associated with resistance to rifampicin, dapsone, and fluoroquinolones, respectively, were detected among these 12 samples. No mutations were either detected in gyrB , ctpC , ctpI , or nth among the samples where sequencing results were available for at least one of these targets, SNP and VNTRs Genotyping In these 12 samples, the SNP A1015G, characteristic of the 1D-Malagasy genotype, was identified via Sanger sequencing (Table 4 ). These strains were thus classified within the 1D-Malagasy phylogenetic subgroup [ 6 ], reflecting likely stable local transmission with limited influence from exogenous strains, as commonly observed in leprosy-endemic foci [ 34 ]. Among these 12 genotype 1D-Malagasy samples, 10/12 were analyzed with the Deeplex Myc-Lep assay and 7 yielded interpretable results for hsp65 , SNP type and a minimum of 9 VNTR loci (6-3a, AC9, AC8b, GAA21, GGT5, AC8a, GTA9, 12 − 5, 23 − 3) (Fig. 4 , Table 4 , Supplementary Table 4). Consistently with the identification by LiPA and Sanger sequencing of ml2446 , all were confirmed as M. leprae and SNP type 1/1D, based on the sequencing results of hsp65 and 18 SNP regions. The Hunter-Gaston discriminatory index (H) of VNTR loci indicated that GAA21 (H = 0.95), GTA9 (H = 0.66), and AC8a (H = 0.56) were the most informative loci for strain differentiation (Table 4 ). AC9 showed moderate variability (H = 0.47), while the remaining loci were non-polymorphic (H = 0) (Table 4 ). Despite the small sample size, these results suggest that GAA21, GTA9, and AC8a are the most useful VNTR loci for distinguishing M. leprae strains in this setting. While their shared 1D-Malagasy SNP type, the samples with an interpretable VNTR patterns all exhibited a different VNTR profile. Information on hsp65 best match–based identification, the VNTR allelic profile, and the SNP-based phylogenetic lineage is displayed in the center of the circular map. Predictions of drug susceptibility or resistance to anti-leprosy drugs and identification of a potential hypermutator genotype are shown in the outer sectors. Target gene regions are grouped into sectors according to the associated prediction feature (drug resistance or hypermutation). Green color in all sectors indicate the absence of resistance- or hypermutation-associated mutations, respectively, resulting in predictions of susceptible phenotypes (for rpoB, folP1, gyrA, and gyrB) or no detection of a hypermutator strain (for nth). The ctpC and ctpI sectors, along with their corresponding resistance or susceptibility predictions, are classified as exploratory. This categorization is based on previous work suggesting a possible association between missense mutations in these genes and rifampicin resistance in a single strain lacking mutations in the rpoB drug-resistance determining region. Green lines above gene names represent reference sequences with coverage breadth > 95%. The limit of detection (LOD) for minority variants arising from subpopulations of sequencing reads carrying a mutation—depends on read depth at each position and is indicated by grey (LOD 10%) or orange zones (LOD > 10%) above the reference sequences. Results were obtained for the full set of 11 core VNTR loci covered by Deeplex Myc-Lep for this sample. VNTR markers are presented in the following order: 6-3a, AC8a, AC8b, AC9, GTA9, GAA21, GGT5, 6–7, 12 − 5, 21 − 3, and 23 − 3. Abbreviations: RIF, rifampicin; DPS, dapsone; FQ, fluoroquinolones; VNTR, variable-number tandem repeat; SNP, single nucleotide polymorphism. Based on multi-locus VNTR patterns, a dendrogram was constructed to assess genetic similarity among samples. This analysis identified three distinct genetic patterns, referred to as Groups A, B, and C (Supplementary Fig. 1B). These groups were distinguished by specific allele combinations, particularly at GAA21, which showed clear differentiation: higher allele values in Group A, intermediate in Group B, and lower in Group C (Table 4 ). Table 4 Repeat polymorphisms of 9 polymorphic VNTR loci with the Hunter-Gaston discriminatory index (H) reported for each locus. ID Village GAA21 GTA9 AC8a AC9 6-3a AC8b GGT5 12 − 5 23 − 3 SNP Subtype Group 9018 Marotongo 28 10 9 8 3 7 5 4 2 1 D-Malagasy A 9013 Ankazondrigintra 21 6 10 8 3 7 5 4 2 1 D-Malagasy A 9009 Ambalakida 17 9 9 8 3 7 5 4 2 1 D-Malagasy B 9023 Andrafiabe 18 9 9 8 3 7 5 4 2 1 D-Malagasy B 9021 Andrafiabe 17 10 10 9 3 7 5 4 2 1 D-Malagasy B 9068 Antsoha 14 10 10 9 3 7 5 4 2 1 D-Malagasy C 9048 Besakoa 15 10 10 8 3 7 5 4 2 1 D-Malagasy C H 0.95 0.66 0.56 0.47 0 0 0 0 0 ID: patient identifier; Village: village of origin of the patients within the Miandrivazo district; VNTR: Variable Number Tandem Repeat (6-3a, AC9, AC8b, GAA21, GGT5, AC8a, GTA9, 12 − 5, 23 − 3), H: Hunter-Gaston discriminatory index; SNP: Single Nucleotide Polymorphism. Group: cluster defined by VNTR analysis. Discussion The PEOPLE study, conducted in Miandrivazo district, identified 80 new leprosy cases among 20,221 individuals who were clinically examined through door-to-door screening over a three-year period. 70 were detected at baseline in 2019, corresponding to a prevalence of 34/10,000 inhabitants, while the remaining cases were identified in 2020 and 2021, with an incidence of 4.95/10,000 inhabitants[ 9 ]. Among them, 75 were undergoing MDT treatment at the time of sampling, while five had not yet been treated, 4 of whom underwent biological sampling. Delays in sample collection were mainly due to COVID-19 related lockdowns, which prevented teams from Antananarivo from traveling to Miandrivazo. Also, even when local staff identified cases, on-site sampling before onset of treatment was not feasible due to the lack of test consumables. Among the 12 molecularly analysable biopsy samples, representing 15% of the cohort, no mutations associated with resistance to rifampicin, dapsone, or fluoroquinolones were detected by any of the three different assays, based either on separate or combined results. These findings indicate that the current treatment regimen in the region remains effective. Furthermore, the sensitivity of circulating strains to rifampicin supports the potential effectiveness of its use as post-exposure prophylaxis among contacts during the PEOPLE study. Our results are similar to those reported in the Comoros, a neighboring country: most strains had the same 1D genotype (supposedly of Malagasy subtype), and M. leprae remained fully sensitive to rifampicin, fluoroquinolones, and dapsone in 216 patients [ 21 ]. However, the limited sample size analyzed in this pilot study warrants caution in interpreting these results, as they may underestimate the true prevalence of drug resistance. Increasing the number of tested samples would strengthen surveillance, especially since rifampicin resistance has already been observed for other diseases in Madagascar, such as tuberculosis (1.3% of patients between 2012 and 2017) [ 35 ]. Globally, resistance to anti-leprosy drugs remains a concern. A meta-analysis of 4,454 patients reported resistance rates of 5.1% for rifampicin, 7.4% for dapsone, 1.8% for clofazimine, and 3.5% for ofloxacin, with regional variations (12.5% in Africa, 7.5% in the Americas, 16.9% in Europe)[ 35 ]. Despite possible bias due to excluded studies, these findings highlight the need for continuous surveillance to detect emerging resistance and maintain MDT effectiveness. In addition to resistance monitoring, molecular characterization of circulating strains provided complementary insights into local transmission dynamics. Strain genotyping showed homogeneity with the exclusive presence of the genotype 1D-Malagasy, consistent with the dominance of this genotype in other Madagascar settings reported by Avanzi et al. [ 6 ]. This homogeneity may reflect a local and persistent circulation of these strains. However, the detection of genotype 1A in two patients from the Boeny region, as reported by Avanzi et al . [ 6 ], suggests that this strain likely also circulates in other regions, potentially including the Menabe areas studied here. Larger studies are needed to complement these existing observations, confirm the distribution of genotypes, and better understand the persistence and transmission of M. leprae strains in Madagascar. Despite the small size of this pilot dataset, VNTR analysis remarkably differentiated all analysable strains sharing the same SNP 1D-Malagasy genotype, with GAA21, GTA9, and AC8a highlighted as highly discriminatory markers. The epidemiological significance of these unique genotypic profiles is supported by the fact that they originated from different neighborhoods and households, with no known contacts between them. These initial observations are consistent with the well-recognized high-resolution power of VNTR typing, making it particularly suited to fine-scale studies of M. leprae transmission over short epidemiological distances. As doable by utilizing the Deeplex Myc-Lep assay, combining SNP and VNTR approaches will therefore be valuable to identify distinct regional clusters within the Malagasy SNP 1D genotype, thereby improving the understanding of M. leprae transmission dynamics and spatial structuring in Madagascar. The RLEP qPCR test proved essential as a triage tool before resistance analysis (LiPA, Sanger, and Deeplex Myc-Lep). A Ct threshold for positivity of < 27 was set to maximize chances of successful results across the three tests, as sensitivity declines beyond this point. In our cohort, which was predominantly composed of patients under treatment, the detection rate of M. leprae by RLEP qPCR from SB was 85%, suggesting that the clinical leprosy diagnosis was overall highly accurate. The result is comparable to those reported in untreated patients by Martinez et al . (87%)[ 15 ] and Azevedo et al. (84.9%) [ 36 ]. RLEP qPCR was highly sensitive for detecting and quantifying M. leprae DNA in both PB and MB cases. However, it does not allow distinction between viable and non-viable bacilli [ 37 ]. Complementary approaches, such as mouse footpad inoculation, RNA-based viability assays [ 33 ], or the morphological index on microscopy, would therefore have been necessary, which, unfortunately, was not feasible in this study. Yet, even after several months of treatment, RLEP qPCR remains sufficiently sensitive to detect and quantify M. leprae DNA in skin lesions. One patient was RLEP qPCR positive (Ct = 16.6) after six months of treatment, based on both SB and NS samples. This patient also was seropositive for anti-PGL-I IgM. However, these results do not allow conclusions regarding bacterial viability or the patient’s infectiousness, as these markers may reflect the presence of non-viable bacilli or bacterial debris. It has been widely reported that IgM levels against M. leprae PGL-I strongly correlate with bacterial load in both humans and animals[ 38 , 39 , 19 ]. Furthermore, these antibodies are reduced upon efficient treatment of patients.[ 40 , 41 ]. However, anti-PGL-I antibodies do not confer protection against leprosy [ 42 ]. Due to the fact that most patients in this study were sampled only during treatment and not at diagnosis, no statements can be made here other than the PGL-I rapid test using FSB was applicable in the field and that the anti-PGL-I IgM levels inversely correlated with Ct values as previously found [ 29 ]. Finally, the elevated acceptance of NS and FSB samples suggests that these low-invasive methods are well tolerated by leprosy patients in Madagascar. Their simplicity, already widely used for other diseases in the country, contributes to higher acceptance compared to SB, which are slightly uncomfortable and require local anesthetics. These types of samples have been employed in several countries for the detection of M. leprae -infected individuals as well as to identify active cases. FSB samples can enable the detection of immunological biomarkers related to infection and disease, including antibodies, chemokines, cytokines and acute phase proteins. When quantified, the levels of these host biomarkers allow differentiation of various clinical phenotypes of leprosy (MB and PB) from control individuals [ 43 ]. In this context, a study is currently underway to validate several of these host biomarkers in patients from Madagascar. A major limitation of our study is the relatively small number of samples analyzed, with most patients already undergoing anti-leprosy treatment at the time of sampling and the absence of AFB smear results. Conclusions The results from this pilot study suggest that M. leprae strains circulating in the Miandrivazo district in Madagascar are sensitive to the main anti-leprosy drugs. RLEP qPCR was implemented in the reference laboratory to pre-select samples with sufficient DNA levels, defined by a Ct threshold of < 27, which ensured successful results of the three molecular drug-resistance assays utilized. The 1D-Malagasy genotype remains predominant in this district. However, VNTR analysis detected all analyzable strains of this type, suggesting the possibility of extending this approach at a larger regional or national scale to obtain better insights into the circulation and transmission of individual strains. Despite the limited size of this pilot study, these findings provide a solid basis for strengthening drug resistance surveillance and genotyping of M. leprae strains in Madagascar, offering perspectives for more effective leprosy control in the country. Abbreviations WHO (World Health Organization), MB (Multibacillary); PB (Paucibacillary); MDT (Multi-drug therapy); PEP (Post-Exposure Prophylaxis); FSB (Finger stick blood); NS (Nasal swab) SB (Skin biopsy); Ct (Cycle threshold); R (Ratio); UCP-LF (up-converting reporter particles Lateral Flow); (PGL-I – Phenolic Glycolipid-I); IgM (Immunoglobulin M); M. leprae (Mycobacterium leprae); qPCR (Quantitative polymerase chain reaction); RLEP (Repetitive element of M. leprae genome); SNP (Single nucleotide polymorphism); VNTR (Variable number tandem repeat); H (Hunter Gaston diversity index); CERBM (Comité d’Éthique de la Recherche Biomédicale); PEOPLE (Post-Exposure Prophylaxis for Leprosy); LiPA (Line Probe Assay); gyrA ( Gene encoding DNA gyrase subunit A); gyrB ( Gene encoding DNA gyrase subunit B); folP1 ( Gene encoding Dihydropteroate synthase); rpoB – Gene encoding RNA polymerase β subunit). Declarations Ethics approval and consent to participate This study was conducted in accordance with the Declaration of Helsinki (7th revision, 64th meeting, 2013, Fortaleza). Ethical approval was obtained from the “Comité d’Éthique de la Recherche Biomédicale” (CERBM) in Madagascar. The trial was registered in the public ClinicalTrials.gov registry (NCT03662022, September 7, 2018, https://clinicaltrials.gov/ct2/show/NCT03662022). Written informed consent was obtained from each participant or, for participants under 18 years of age, from a parent or guardian. Additional consent was obtained from leprosy patients for biological sample collection. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request Competing interests We declare no conflict of interest. Funding This study is part of the PEOPLE project, which is embedded in the EDCTP2 programme and funded by the European Union (grant number RIA2017NIM-1847-PEOPLE). The views and opinions expressed in this publication are solely those of the authors and do not necessarily reflect those of EDCTP. Authors' contributions Conceptualization: BCD, EC, BC, MA, PC, EH, AG. Data curation: LM, SR, TR, AJ, PS. Formal analysis: LM, SR, BC, JLB, SB, AJ, PS, AG. Funding acquisition: BCD, EC, MA, EH, LHS, BC, AG. Methodology: SR, LM, SMB, DJ, PS, BCD, EC, AG. Sample collection: SR, LM, LMA, AR, BC. Supervision: BC, SR, SMB, TM, EC, BCD, AG. Visualization: LM, JLB, AG. Writing original draft: LM, SR, JLB, TR, AG. Writing review and editing: LM, SR, SMB, PC, DJ, AR, LMA, MA, JLB, EC, AJ, PS, EH, BCD, BC, TR, LHS, AG. All authors read and approved the final manuscript. Acknowledgements We express our sincere gratitude to the medical teams on the ground for their dedication to the door-to-door active screening and sample collection. We also thank the teams at NLP Madagascar and the Raoul Follereau Foundation for their support. Finally, we extend our sincere appreciation to all participants and patients who contributed to this study. Authors' information (optional) 1 Centre d’Infectiologie Charles Mérieux, Madagascar (CICM), 2 Dept of Leiden University Center of Infectious Diseases (LUCID), Leiden University Medical Center, Leiden, the Netherlands, 3 Raoul Follereau Foundation (FRF), Antananarivo, Madagascar, 4 Institute of Tropical Medicine (IMT), Antwerp, Belgium, 5 Dept. of Cel and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands, 6 National Leprosy Program (NLP), Madagascar, 7 Fondation Mérieux Lyon, 8 INSERM, IAME UMR1137, Université de Paris Cité 75018 Paris, France, 9 Univ. 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Supplementary Files SupplementaryfileAssessingimprovedsurveillanceofleprosydrugresistanceinMadagascarApilotstudyintheMiandrivazodistrict.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 29 Apr, 2026 Reviews received at journal 19 Apr, 2026 Reviewers agreed at journal 16 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Submission checks completed at journal 07 Apr, 2026 First submitted to journal 06 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019–UMR 9017, Center for Infection and Immunity of Lille","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Supply","suffix":""},{"id":625738650,"identity":"767e3f82-2b3b-4319-88ff-9870476af0ed","order_by":11,"name":"Agathe Jouet","email":"","orcid":"","institution":"Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019–UMR 9017, Center for Infection and Immunity of Lille","correspondingAuthor":false,"prefix":"","firstName":"Agathe","middleName":"","lastName":"Jouet","suffix":""},{"id":625738651,"identity":"7b43ff24-1b52-4d00-b689-535f11aa1516","order_by":12,"name":"Epco Hasker","email":"","orcid":"","institution":"Institute of Tropical Medicine Antwerp","correspondingAuthor":false,"prefix":"","firstName":"Epco","middleName":"","lastName":"Hasker","suffix":""},{"id":625738652,"identity":"f376e973-6f55-42dc-aae2-f60c38e3729c","order_by":13,"name":"Bouke Catherine de Jong","email":"","orcid":"","institution":"Institute of Tropical Medicine Antwerp","correspondingAuthor":false,"prefix":"","firstName":"Bouke","middleName":"Catherine","lastName":"de Jong","suffix":""},{"id":625738654,"identity":"1218b109-9874-44f4-afe9-b44814d504be","order_by":14,"name":"Bertrand Cauchoix","email":"","orcid":"","institution":"Raoul Follereau Foundation","correspondingAuthor":false,"prefix":"","firstName":"Bertrand","middleName":"","lastName":"Cauchoix","suffix":""},{"id":625738655,"identity":"9ce87dbb-fd7d-4d4d-93d0-0058bed8a69a","order_by":15,"name":"Tahinamandranto Rasamoelina","email":"","orcid":"","institution":"Centre d’Infectiologie Charles Mérieux","correspondingAuthor":false,"prefix":"","firstName":"Tahinamandranto","middleName":"","lastName":"Rasamoelina","suffix":""},{"id":625738656,"identity":"beb2f369-9c3f-4208-ba59-f2eb7207b297","order_by":16,"name":"Luc Hervé Samison","email":"","orcid":"","institution":"Centre d’Infectiologie Charles Mérieux","correspondingAuthor":false,"prefix":"","firstName":"Luc","middleName":"Hervé","lastName":"Samison","suffix":""},{"id":625738658,"identity":"151bf73c-5fb7-40ae-adb6-25f231056763","order_by":17,"name":"Annemieke Geluk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYLCCBwwJcnwQpgWRWhIYEozZIEwJ4rUkthGthb+B/eKDxLa09DaJ7MQPjDuI0CJxgKfYILEtJ7dNInezBOMZYhx2gCdNIrGtAqRlGwNjGxFa5A/wpP8AaklnI1qLwQH2YwxAhyUQr8XwMA+zRMK5NMM2nrebgS4kQovc8faHHz6UJcvzs+du/PCxzYawFgZmHgMEJ4EIDUDA/oA4daNgFIyCUTByAQD5GTL+qfRR+gAAAABJRU5ErkJggg==","orcid":"","institution":"Leiden University Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Annemieke","middleName":"","lastName":"Geluk","suffix":""}],"badges":[],"createdAt":"2026-04-06 15:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9335540/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9335540/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107450238,"identity":"d1917d40-4249-405e-9d55-9257bdd14e0d","added_by":"auto","created_at":"2026-04-21 15:11:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":231678,"visible":true,"origin":"","legend":"\u003cp\u003eStudy workflow\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9335540/v1/0133bffa0450998043fbc93b.png"},{"id":107450229,"identity":"559bbfca-60a3-479a-879f-878b96e60ff7","added_by":"auto","created_at":"2026-04-21 15:11:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82134,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of acceptance rates across sampling methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e – Percentage of acceptance for sampling of all sample types; \u003cstrong\u003eB\u003c/strong\u003e – Acceptance rate by sample type among 62 consenting patients. SB: Skin biopsy (n=33), NS: nasal swab (n=57), FSB: Fingerstick blood (n=47).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9335540/v1/c07247ab4f435880a4b8b256.png"},{"id":107450200,"identity":"2b155c11-7f14-4a8d-8c74-53199199357c","added_by":"auto","created_at":"2026-04-21 15:11:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":33056,"visible":true,"origin":"","legend":"\u003cp\u003eDirect and indirect diagnostics for paucibacillary (PB) and multibacillary (MB)\u003cstrong\u003e \u003c/strong\u003epatients\u003cstrong\u003e (A)\u003c/strong\u003e Ct values from RLEP qPCR of skin biopsies in PB and MB patients. Red dots represent patients who are seropositive for anti-PGL-I IgM antibodies. \u003cstrong\u003e(B)\u003c/strong\u003eOutcome measures of the anti-PGL-I test in FSB, expressed as the ratio R.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9335540/v1/14ddd10c8ab23417c271f405.png"},{"id":107450220,"identity":"57f39644-34c0-4748-a7b4-4ee52bf06147","added_by":"auto","created_at":"2026-04-21 15:11:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":137529,"visible":true,"origin":"","legend":"\u003cp\u003eDeeplex Myc-Lep results identifying a \u003cem\u003eM. leprae\u003c/em\u003e strain of SNP type 1D genotypically susceptible to rifampicin, dapsone and fluoroquinolones in sample 9013, from a patient with leprosy from the Miandrivazo district, Madagascar.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9335540/v1/4038cc3de3433ac692f47d57.png"},{"id":107490240,"identity":"6fc8917d-fa8c-4850-8a15-43e80a4769d2","added_by":"auto","created_at":"2026-04-22 02:51:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1055590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9335540/v1/15f3b234-ca3b-441e-b31c-4c402b74de12.pdf"},{"id":107450224,"identity":"7acedb3a-1f5a-4ae8-bc70-3d1dd50e191a","added_by":"auto","created_at":"2026-04-21 15:11:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":217922,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileAssessingimprovedsurveillanceofleprosydrugresistanceinMadagascarApilotstudyintheMiandrivazodistrict.docx","url":"https://assets-eu.researchsquare.com/files/rs-9335540/v1/96e6ef26ad2ad03c156af4fc.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessing improved surveillance of leprosy drug resistance in Madagascar: A pilot study in the Miandrivazo district","fulltext":[{"header":"Background","content":"\u003cp\u003eLeprosy, caused by \u003cem\u003eMycobacterium leprae\u003c/em\u003e or \u003cem\u003eM. lepromatosis\u003c/em\u003e, is a chronic granulomatous disease primarily affecting the skin, peripheral nerves, and mucous membranes. The introduction of multidrug therapy (MDT), combining dapsone, rifampicin, and clofazimine, has significantly reduced the global prevalence of leprosy. However, over the past ten years, the average annual number of new cases has remained stable, with 172,717 new cases and 4,488 relapses reported in 2024 across more than 188 countries, reflecting the persistence of transmission. Furthermore, the emergence of drug-resistant leprosy has been reported in several endemic countries, prompting the World Health Organization (WHO) to recommend vigilant monitoring of resistance to key anti-leprosy medications, particularly among multibacillary (MB) patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The 2009\u0026ndash;2015 surveillance of 1932 cases of anti-leprosy drug resistance (1143 relapse and 789 new cases) in 19 countries reported that 8.0% of \u003cem\u003eM. leprae\u003c/em\u003e strains were resistant to anti-leprosy drugs. Regarding rifampicin, resistance was observed in Brazil (\u0026asymp;\u0026thinsp;9.1%), India (\u0026asymp;\u0026thinsp;4.7%), Colombia (\u0026asymp;\u0026thinsp;24.3%), and Indonesia (\u0026asymp;\u0026thinsp;3.3%) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Madagascar, approximately 1,500 new cases are still detected annually, of whom 10% are children under 15 years old, indicating ongoing transmission [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In 2021, 1,340 patients received treatment, highlighting the persistent disease burden [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In 2015 the Centre d\u0026rsquo;Infectiologie Charles M\u0026eacute;rieux (CICM), the national reference laboratory for leprosy, began molecular monitoring of anti-leprosy drug resistance, in collaboration with both the national leprosy program and international partners. This surveillance was initially focused in Antananarivo and some leprosy endemic regions, thereby reducing the geographical representation of the data gathered. Currently, only one case of drug resistance has been recorded, based on the screening of high-risk patients recommended by clinicians (approximately 5\u0026ndash;10 cases per year), which may indicate insufficient testing coverage rather than an actual lack of resistant strains [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, genotyping studies of \u003cem\u003eM. leprae\u003c/em\u003e strains have identified the 1D-Malagasy genotype, defined by the A1015G SNP in the gene \u003cem\u003eml2446\u003c/em\u003e encoding the lipoprotein Q (LprQ), as the hyperdominant strain type circulating in these settings [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nevertheless, among the 30 samples analyzed in that study, only two originated from the Menabe region, which includes the Miandrivazo district, a hyperendemic area for leprosy in western Madagascar.\u003c/p\u003e \u003cp\u003eFrom 2018\u0026ndash;2023, Madagascar participated in the PEOPLE study (Post-Exposure Prophylaxis for Leprosy), a randomized clinical trial (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clinicaltrials.gov/ct2/show/NCT03662022\u003c/span\u003e\u003cspan address=\"https://clinicaltrials.gov/ct2/show/NCT03662022\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) designed to evaluate the efficacy of different post-exposure prophylaxis (PEP) strategies against leprosy. In Madagascar, the Miandrivazo district was selected as the study site based on its high incidence, to assess the effectiveness of single dose of rifampicin (SDR) administered prophylactically to contacts of leprosy patients [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This study has allowed identification of both new and previously diagnosed leprosy cases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], providing a unique opportunity to assess resistance to anti-leprosy drugs in this area, particularly rifampicin, the drug endorsed by WHO for post-exposure prophylaxis. Given that rifampicin is applied in tuberculosis (TB) and leprosy health care, both for treatment and prophylaxis, it is important to survey the presence of resistant strains to prevent their selection or spread [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince \u003cem\u003eM. leprae\u003c/em\u003e cannot be cultured \u003cem\u003ein vitro\u003c/em\u003e and phenotyping based on culture in the mouse footpad model is lengthy and costly, molecular tests have been developed to directly detect, from clinical samples, mutations associated with antimicrobial resistance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To select samples for resistance testing, the WHO recommends prioritizing MB patients with bacteriological index (BI)\u0026thinsp;\u0026ge;\u0026thinsp;2, as these samples are more likely to be PCR-positive [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A BI\u0026thinsp;\u0026ge;\u0026thinsp;2 corresponds approximately to the minimal threshold of 10⁴ bacilli per gram of tissue required for reliable detection of acid-fast bacilli (AFB) by conventional microscopy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Consequently, this method exhibits limited sensitivity, and by definition negative in paucibacillary (PB) patients. In contrast, the RLEP qPCR assay stands out as a sensitive molecular tool capable of identifying \u003cem\u003eM. leprae\u003c/em\u003e DNA, even in samples with low bacterial counts [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Cycle threshold (Ct) values from RLEP qPCR also enable precise quantification of \u003cem\u003eM. leprae\u003c/em\u003e genome copy numbers in the DNA extracts. Because sufficient bacterial DNA is required for molecular drug-resistance analyses, RLEP qPCR facilitates the identification of samples eligible for detection of possible drug resistance-associated mutations.\u003c/p\u003e \u003cp\u003eMolecular testing of resistance of \u003cem\u003eM. leprae\u003c/em\u003e to rifampicin, dapsone and fluoroquinolones is based on known resistance mutations in regions of the \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003efolP1\u003c/em\u003e, and \u003cem\u003egyrA/gyrB\u003c/em\u003e genes, respectively. Available methods include the GenoType Leprae DR (Hain Lifescience/Bruker) line probe assay based on post-PCR reverse hybridization [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], targeted Sanger sequencing [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and the recently developed targeted next-generation sequencing Deeplex-Myc-Lep assay (GenoScreen)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition to resistance testing and identification of \u003cem\u003eM. leprae\u003c/em\u003e (and \u003cem\u003eM. lepromatosis\u003c/em\u003e), this assay simultaneously provides high-resolution genotyping based on multiple canonical typing SNPs and variable number of tandem repeat (VNTR) loci. Deeplex Myc-Lep has successfully been used to conduct a first countrywide drug resistance survey in the frame of the PEOPLE study, in the Comoros[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to drug resistance detection, the PEOPLE study provides, for the first time in Madagascar, an opportunity to compare acceptance rates of low-invasive sampling methods, including nasal swabs (NS) and finger stick blood (FSB), with skin biopsies (SB) among leprosy patients. RLEP qPCR performed on NS allows for the detection of bacterial colonization, while FSB enables the measurement of anti-\u003cem\u003eM. leprae\u003c/em\u003e PGL-I (phenolic glycolipid I) IgM, a biomarker specific for past and present \u003cem\u003eM. leprae\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eThe anti-PGL-I UCP-LFA test, used to quantify anti-\u003cem\u003eM. leprae\u003c/em\u003e PGL-I IgM from minimally invasive capillary blood samples (FSB), was applied to 1,815 contacts of leprosy patients in our cohort. Analyses revealed a marked reduction in the seroprevalence of anti-PGL-I IgM, decreasing from 27.4% to 11.6% between the first and third year following the prophylactic intervention [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In leprosy patients, it has also been widely demonstrated that IgM levels directed against the PGL-I of \u003cem\u003eM. leprae\u003c/em\u003e are strongly correlated with bacterial load and generally decrease significantly after effective treatment [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This biological sub-study of the PEOPLE project aimed to (i) determine the genetic diversity of the identified \u003cem\u003eM. leprae\u003c/em\u003e strains isolated from the patients using VNTR and SNP analyses after RLEP qPCR-based selection (ii) examine the presence of resistance-associated mutations in target gene regions using LiPA, targeted Sanger sequencing and Deeplex Myc-Lep (iii) evaluate and compare the acceptance rate of low-invasive sampling (NS and FSB) with that of SB and (iv) asses seropositivity for anti-\u003cem\u003eM. leprae\u003c/em\u003e PGL-I IgM in leprosy patients from the Miandrivazo district in Madagascar.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population and clinical samples\u003c/h2\u003e \u003cp\u003eClinical samples and associated patient data were obtained from the PEOPLE study conducted in the Miandrivazo district. Patient screening was performed through a door-to-door approach in the community. The study was implemented in three rural communes in the southern part of the district Ankotrofotsy, Manambina, and Ambatolahy selected for their high endemicity, with an annual prevalence estimated at 1.5 cases per 10,000 population in 2018. Leprosy diagnosis followed the criteria of the WHO: patients presenting 1\u0026ndash;5 skin lesions were classified as PB, and those with more than 5 lesions as MB [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSampling\u003c/h3\u003e\n\u003cp\u003eSB, NS, and FSB samples were collected from all consenting leprosy patients between 0 and 10 months after MDT initiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNS samples were collected using ESWAB\u0026reg; kits (COPAN Italia, Cat. 013708), in accordance with the manufacturer's recommendations. SB were performed using a 4 mm punch after local anesthesia and placed in a microtube containing 1 mL of Disolol. However, SB were not performed in patients with neural forms. For FSB, 20 \u0026micro;l of blood were collected following the detailed protocols described in a previous study [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Timely sampling of all clinically confirmed cases was impeded by seasonal flooding and COVID-19 restrictions in 2020\u0026ndash;2021, limiting field activities to a few dry-season periods between 2019 and 2021. All samples were promptly stored in a cold box in the field (4\u0026ndash;10\u0026deg;C) and subsequently kept at 4\u0026deg;C at Miandrivazo hospital until transportation by road in a refrigerated box (4\u0026ndash;8\u0026deg;C) to the National Reference Laboratory for Leprosy at the CICM in Antananarivo for analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eClinical diagnosis was performed based on WHO classification. Upon obtaining patient consent, samples were collected using skin biopsy and/or nasal swab and/or fingerstick blood (FSB). RLEP qPCR was used to detect \u003cem\u003eM. leprae\u003c/em\u003e in skin biopsies and nasal swabs. The UCP-LFA was used to quantify anti-PGL-I IgM antibodies in FSB samples. 26/28 \u003cem\u003eM. leprae\u003c/em\u003e positive skin biopsy samples were tested for drug resistance and strain genotype (2 samples excluded due to insufficient volume). Among the 26 samples, 12 yielded interpretable results using at least one of 3 techniques. The 14 failed (undetermined) results indicate Ct\u0026thinsp;\u0026gt;\u0026thinsp;27.\u003c/p\u003e\n\u003ch3\u003eNasal swab DNA extraction\u003c/h3\u003e\n\u003cp\u003eDNA extraction was performed using thermal shock with 200 \u0026micro;L of the sample. The inactivation step was carried out at 99\u0026deg;C for 30 minutes in a thermomixer (Eppendorf, Cat. 5355), followed by the addition of 1 \u0026micro;L of a pre-optimized Sample Processing Control (SPC) ([Yakima Yellow\u0026reg;-BHQ-1\u0026reg; Probe] 200 RXN, Eurogentec, RT-SPCY-B02) diluted at 1:10. Then, a thermal shock cycle at 96\u0026deg;C for 2 minutes was applied, followed by cooling on ice for 2 minutes, repeated five times [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Finally, the sample was incubated at 96\u0026deg;C for 15 minutes. And after a brief centrifugation of one minute, the supernatant containing the extracted DNA was recovered and then stored at -20\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eSkin biopsy DNA extraction\u003c/h3\u003e\n\u003cp\u003eBiopsy was finely fragmented and transferred into a tube GentleMACS M containing 500 \u0026micro;L of phosphate-buffered saline (PBS, Sigma, Cat. P4417) and 1 \u0026micro;L of a pre-optimized SPC diluted at 1:10 (from the qPCR DNA extraction and inhibition control kit [Yakima Yellow\u0026reg;-BHQ-1\u0026reg; Probe] 200 RXN, Eurogentec, RT-SPCY-B02). Tissue homogenization was performed using the gentleMACS dissociator (MACS-Miltenyi Biotec) with the RNA_02 program, followed by centrifugation at 2000 rcf for 5 minutes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Subsequently, 200 \u0026micro;L of the homogenate was subjected to chemical lysis and DNA purification using the QIAamp UCP-Pathogen DNA MiniKit, following the manufacturer\u0026rsquo;s protocol (QIAGEN, Cat. 50214). The purified DNA was eluted in 100 \u0026micro;L of TE buffer and stored at -20\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eRLEP qPCR assays\u003c/h3\u003e\n\u003cp\u003eAll DNA extracts from biopsy samples and NS were subjected to quantitative PCR (qPCR) targeting the RLEP repeat sequence to detect and quantify \u003cem\u003eM. leprae\u003c/em\u003e, as previously described, with minor modifications [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The qPCR reaction was performed in a final reaction volume of 20 \u0026micro;l, containing 1X SensiMix II No-Rox (Bioline, Cat. Bio-83005), 400 nM each of forward and reverse primer, 100 nM of the probe, 300 nM ROX (to improve signal stability), 2X EGT control mix (Eurogentec, Cat. RT-IPCY-B02), and 2 \u0026micro;L of purified extracted DNA.\u003c/p\u003e \u003cp\u003eAmplification was performed in a Bio-Rad CFX96 thermocycler, starting with an initial denaturation at 95\u0026deg;C for 10 minutes, followed by 45 cycles of 95\u0026deg;C for 10 seconds and 60\u0026deg;C for 60 seconds, with a final step at 4\u0026deg;C for 1 minute. Each sample was analyzed in analytical triplicate following published protocols [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor each qPCR run, nuclease-free water was included as a contamination control, along with a serial dilution of the \u003cem\u003eM. leprae\u003c/em\u003e reference strain NHDP (3 \u0026times; 10⁶ to 30 RLEP copies) (BEI, Ref. No. 19350). Based on the Ct value, the slope of the regression line, and the Y-intercept, the Bio-Rad CFX96 software automatically calculated the RLEP copy number.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnti-\u003c/b\u003e \u003cb\u003eM. leprae\u003c/b\u003e \u003cb\u003ePGL-I IgM UCP-LFA\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSerological analysis of FSB collected in the field was performed using lateral flow assay (LFA) strips with integrated luminescent upconverting reporter particles (UCP) to quantitatively detect anti-\u003cem\u003eM. leprae\u003c/em\u003e PGL-I IgM, following previously described protocols [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The results are expressed as normalized values (Ratio (R)), equivalent to the ratio between the fluorescence signal measured at the Test line and at the Flow Control line on the UCP-LFA test strip.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLine Probe Assay\u003c/h2\u003e \u003cp\u003eAll \u003cem\u003eM. leprae\u003c/em\u003e-positive samples (RLEP Ct\u0026thinsp;\u0026lt;\u0026thinsp;36) were tested using the GenoType Leprae DR test (Hain Lifescience, IFU-320-06), following the manufacturer\u0026rsquo;s instructions. The target regions included the rifampicin resistance-determining region in \u003cem\u003erpoB\u003c/em\u003e from codon 432 to 458, the quinolone resistance-determining region in \u003cem\u003egyrA\u003c/em\u003e from codon 89 to 91, and the dapsone resistance-determining region in \u003cem\u003efolP1\u003c/em\u003e from codon 53 to 55 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSanger sequencing\u003c/h3\u003e\n\u003cp\u003eSanger sequencing was used to confirm the Line Probe Assay results and to target the specific 1D-Malagasy genotype. We used the primers recommended by the WHO to detect mutations in the \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003egyrA\u003c/em\u003e, and \u003cem\u003efolP1\u003c/em\u003e genes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. For the identification of the specific 1D-Malagasy locus (\u003cem\u003eml2446\u003c/em\u003e), associated with the A1015G mutation, we used the primers described by Avanzi \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Amplification of the target genes was performed following the protocol of Avanzi \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], with minor modifications. The PCR reaction was carried out using TopTaq Master Mix Kit (Qiagen, Cat.200403), and the amplicons were sequenced by the Sanger platform at GenoScreen (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genoscreen.fr/fr/\u003c/span\u003e\u003cspan address=\"https://www.genoscreen.fr/fr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Results were analyzed using MEGA v7.1 software, comparing the detected mutations with those known to confer antibiotic resistance, as described by the WHO for the three studied genes.\u003c/p\u003e\n\u003ch3\u003eDeeplex Myc-Lep\u003c/h3\u003e\n\u003cp\u003eFor the Deeplex\u0026reg; Myc-Lep test, the 10 samples with a RLEP Ct\u0026thinsp;\u0026lt;\u0026thinsp;30 were sent to the \u0026ldquo;Mycobacteriology Service at Bichat-Claude Bernard Hospital in France\u0026rdquo;, where laboratory analyses were conducted in strict accordance with the manufacturer\u0026rsquo;s recommendations, as previously described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The Deeplex\u0026reg; Myc-Lep assay (GenoScreen, GNSRUOLEP01-20) relies on deep sequencing of a single 44-plex amplicon mix for the simultaneous identification of mycobacterial species (based on the \u003cem\u003ehsp65\u003c/em\u003e gene sequence), genotyping (based on SNPs in 18 gene regions and 11 VNTR markers), and prediction of \u003cem\u003eM. leprae\u003c/em\u003e drug resistance. Resistance prediction is based on the detection of known resistance-associated mutations in seven genes: \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003ectpC\u003c/em\u003e, and \u003cem\u003ectpI\u003c/em\u003e (rifampicin); \u003cem\u003efolP1\u003c/em\u003e (dapsone); and \u003cem\u003egyrA\u003c/em\u003e, \u003cem\u003egyrB\u003c/em\u003e (fluoroquinolones) and \u003cem\u003enth\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. VNTR profiles were analyzed with R, visualized with a heatmap, and grouped by hierarchical clustering using Euclidean distance and the UPGMA method.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eCohort characteristics and sampling\u003c/h2\u003e\n \u003cp\u003eOverall, 80 patients presenting clinical signs of leprosy were included, comprising 41 MB and 39 PB cases (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDemographic and clinical information of leprosy patients.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003ePatient characteristics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003ebefore treatment\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eduring treatment\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;75)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003etotal\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;80)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\n \u003cp\u003egender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003emale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003efemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\n \u003cp\u003eage (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e16\u0026ndash;30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e31\u0026ndash;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\n \u003cp\u003eWHO classification\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003ePB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\n \u003cp\u003esamples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eSB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eFSB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003en indicates the number of patients. MB: multibacillary; PB: paucibacillary.; SB: skin biopsies; NS: nasal swab; FSB: finger stick blood.\u003c/p\u003e\n \u003cp\u003eThe cohort was predominantly male 55/80 with a mean age of 41 years, including six children under 16 years. Among the newly diagnosed cases, 5/80 were not under treatment at the time they were sampled, while 75/80 were already receiving MDT for 3 to 10 months (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eRegarding sample collection 62/80 patients agreed to provide at least one sample of SB, FSB, or NS (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 33/80 patients (41%) consented to SB, 57/80 (71%) to NS sampling, and 47/80 (59%) to FSB sampling (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Acceptance of all three samples simultaneously (SB, NS, and FSB) was 41% (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eMoreover, among the 80 patients, 25 (31%) presented with pure neural leprosy (17 neuro-MB, 8 neuro-PB). Acceptance rates were 56% (14/25) for FSB, 84% (21/25) for NS, and 8% (2/25) for SB (Supplementary Table\u0026nbsp;5). The SB was performed only in two patients with pure neural leprosy without skin lesions, for whom nerve-level SB above the knee were conducted with patient consent, in accordance with national guidelines.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eRLEP qPCR for skin biopsies\u003c/h2\u003e\n \u003cp\u003eAmong the 33 patients who consented to SB sampling, 10/13 PB patients and 18/20 MB patients tested positive by RLEP qPCR (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), yielding an overall positivity rate of 85%. The median Ct values were 30.0 (IQR 24.0\u0026ndash;32.1) for MB patients and 32.0 (IQR 30.9\u0026ndash;33.7) for PB patients. SB RLEP qPCR results demonstrated that MB patients carried significantly higher concentrations of \u003cem\u003eM. leprae\u003c/em\u003e DNA than PB patients (p\u0026thinsp;=\u0026thinsp;0.03) (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), consistent with the expected relationship between bacterial load and clinical form.\u003c/p\u003e\n \u003cp\u003eAnalysis of bacterial load over treatment showed that Ct values exhibited no significant temporal trend in PB patients (nptrend z = \u0026minus;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.96) or MB patients (nptrend z\u0026thinsp;=\u0026thinsp;1.03, p\u0026thinsp;=\u0026thinsp;0.30), although values for MB tended to increase with treatment duration (Supplementary Table 3). These results showed the high sensitivity of RLEP qPCR for detecting \u003cem\u003eM. leprae\u003c/em\u003e DNA during and after MDT, confirming leprosy before treatment in one PB and three MB patients. However, a DNA-based PCR test cannot distinguish viable from non-viable bacilli, particularly during the first months of MDT when \u003cem\u003eM. leprae\u003c/em\u003e is rapidly killed (\u0026lt;\u0026thinsp;3 months), yet DNA can linger for years [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e: Diagnostic test data for treated and untreated leprosy patients.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003ePatients classified as multibacillary (MB) and paucibacillary (PB), and according to treatment status at the time of sampling (untreated vs treated for 3 to 10 months). N: total number of patients; n: number of patients tested per technique. Results are expressed as the number of positives and negatives for \u003cem\u003eMycobacterium leprae\u003c/em\u003e detection by RLEP qPCR skin biopsies (SB), nasal swabs (NS) and as seropositive/seronegative for anti-PGL-I IgM by finger stick blood (FSB).\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eRLEP qPCR RLEP for nasal swabs\u003c/h2\u003e\n \u003cp\u003eRLEP qPCR was positive in 4 of the 57 patients who consented to NS sampling, all of whom had MB leprosy with Ct values of 27.78, 28.96, 33.35, and 31.93. These patients had highest skin bacterial loads with Ct values of 14.01, 14.27, 16.6, and 25.32 in the RLEP qPCR performed on the concurrent SB samples (Supplementary Table 3). These results suggest that the detection of \u003cem\u003eM. leprae\u003c/em\u003e in NS is primarily observed in MB patients with a high bacterial load as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] .\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eAnti-PGL-I IgM levels in FSB\u003c/h2\u003e\n \u003cp\u003eAmong the 47 patients who consented to FSB sampling (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), only one had not yet started MDT at the time of sampling and was seronegative for anti-PGL-I IgM.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDemographic, clinical, and diagnostic data of six patients highly seropositive for anti-PGL-I IgM.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eMonths of MDT before sampling\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eClinical signs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003eCt SB\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003eRLEP\u003c/p\u003e\n \u003cp\u003eqPCR SB\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003eCt NS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003eRLEP\u003c/p\u003e\n \u003cp\u003eqPCR NS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003ePGL-I IgM (R)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003edMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e16.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003epos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e33.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003epos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\n \u003cp\u003e3.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9073\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003enMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\n \u003cp\u003e1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003edMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e27.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003epos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003edMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003edMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e29.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003enPB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003eneg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eNA: not applicable (no samples available as patients did not give consent); d: dermatological; n: neural; MB: multibacillary; PB: paucibacillary; neg: negative; pos: positive; Ct: cycle threshold; ND: not determined; M: male; NS: nasal swab; SB: skin biopsy; R: ratio between the fluorescence signal measured at the test line and at the flow control line on the UCP-LFA test strip.\u003c/p\u003e\n \u003cp\u003eThe other 46 were undergoing MDT treatment at the time of collection (Supplementary Table 3), of whom six (1 PB; 5 MB) were seropositive for anti-PGL-I IgM (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Among these 6 seropositive patients, 4 were detected positive for \u003cem\u003eM. leprae\u003c/em\u003e by RLEP qPCR on SB samples. The two remaining patients are a neuro-MB and neuro- PB, for whom no SB could be performed. They were 42 and 38 years old, respectively, both at the 6th month of MDT, with anti-PGL-I IgM levels (R value) of 0.97 and 0.36 (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe FSB sample from MB patient, aged 32 years, presented an high R-value of 3.59 and displayed Ct values of 16.6 for the SB. and 33.34 for the NS in RLEP qPCR (Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which is in line with previous findings [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] which reported a correlation between molecular and serological test result.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eDrug resistance\u003c/h2\u003e\n \u003cp\u003eRLEP qPCR was used as a pre-screening test for further molecular antibiotic resistance analyses, depending on Ct values, which were performed using three complementary techniques: LiPA, Sanger sequencing, and Deeplex Myc-Lep. Among the 26 samples tested with all three methods, 6/26 (with a Ct\u0026thinsp;\u0026lt;\u0026thinsp;27) yielded interpretable results across all techniques (Supplementary Table\u0026nbsp;4). Three samples provided interpretable results only with Deeplex Myc-Lep, while three others, analyzed by LiPA and Sanger sequencing but not submitted to Deeplex Myc-Lep due to a Ct above the recommended threshold (Ct\u0026thinsp;\u0026lt;\u0026thinsp;27), also produced usable results. Performance decreased markedly above Ct of 27, particularly for LiPA and Sanger sequencing, likely due to a too low bacillary load.\u003c/p\u003e\n \u003cp\u003eOf the 26 DNA samples extracted from SB, 12 yielded interpretable results using at least one of the employed techniques (Supplementary Table 4). No mutations (Deeplex Myc-Lep and Sanger sequencing) or no known mutations (LiPA) in the \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003efolP1\u003c/em\u003e, or \u003cem\u003egyrA\u003c/em\u003e genes associated with resistance to rifampicin, dapsone, and fluoroquinolones, respectively, were detected among these 12 samples. No mutations were either detected in \u003cem\u003egyrB\u003c/em\u003e, \u003cem\u003ectpC\u003c/em\u003e, \u003cem\u003ectpI\u003c/em\u003e, or \u003cem\u003enth\u003c/em\u003e among the samples where sequencing results were available for at least one of these targets,\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eSNP and VNTRs Genotyping\u003c/h2\u003e\n \u003cp\u003eIn these 12 samples, the SNP A1015G, characteristic of the 1D-Malagasy genotype, was identified via Sanger sequencing (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These strains were thus classified within the 1D-Malagasy phylogenetic subgroup [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], reflecting likely stable local transmission with limited influence from exogenous strains, as commonly observed in leprosy-endemic foci [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eAmong these 12 genotype 1D-Malagasy samples, 10/12 were analyzed with the Deeplex Myc-Lep assay and 7 yielded interpretable results for \u003cem\u003ehsp65\u003c/em\u003e, SNP type and a minimum of 9 VNTR loci (6-3a, AC9, AC8b, GAA21, GGT5, AC8a, GTA9, 12\u0026thinsp;\u0026minus;\u0026thinsp;5, 23\u0026thinsp;\u0026minus;\u0026thinsp;3) (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Table 4). Consistently with the identification by LiPA and Sanger sequencing of \u003cem\u003eml2446\u003c/em\u003e, all were confirmed as \u003cem\u003eM. leprae\u003c/em\u003e and SNP type 1/1D, based on the sequencing results of \u003cem\u003ehsp65\u003c/em\u003e and 18 SNP regions. The Hunter-Gaston discriminatory index (H) of VNTR loci indicated that GAA21 (H\u0026thinsp;=\u0026thinsp;0.95), GTA9 (H\u0026thinsp;=\u0026thinsp;0.66), and AC8a (H\u0026thinsp;=\u0026thinsp;0.56) were the most informative loci for strain differentiation (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). AC9 showed moderate variability (H\u0026thinsp;=\u0026thinsp;0.47), while the remaining loci were non-polymorphic (H\u0026thinsp;=\u0026thinsp;0) (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eDespite the small sample size, these results suggest that GAA21, GTA9, and AC8a are the most useful VNTR loci for distinguishing \u003cem\u003eM. leprae\u003c/em\u003e strains in this setting. While their shared 1D-Malagasy SNP type, the samples with an interpretable VNTR patterns all exhibited a different VNTR profile.\u003c/p\u003e\n \u003cp\u003eInformation on hsp65 best match\u0026ndash;based identification, the VNTR allelic profile, and the SNP-based phylogenetic lineage is displayed in the center of the circular map. Predictions of drug susceptibility or resistance to anti-leprosy drugs and identification of a potential hypermutator genotype are shown in the outer sectors. Target gene regions are grouped into sectors according to the associated prediction feature (drug resistance or hypermutation). Green color in all sectors indicate the absence of resistance- or hypermutation-associated mutations, respectively, resulting in predictions of susceptible phenotypes (for rpoB, folP1, gyrA, and gyrB) or no detection of a hypermutator strain (for nth). The ctpC and ctpI sectors, along with their corresponding resistance or susceptibility predictions, are classified as exploratory. This categorization is based on previous work suggesting a possible association between missense mutations in these genes and rifampicin resistance in a single strain lacking mutations in the rpoB drug-resistance determining region. Green lines above gene names represent reference sequences with coverage breadth\u0026thinsp;\u0026gt;\u0026thinsp;95%. The limit of detection (LOD) for minority variants arising from subpopulations of sequencing reads carrying a mutation\u0026mdash;depends on read depth at each position and is indicated by grey (LOD 10%) or orange zones (LOD\u0026thinsp;\u0026gt;\u0026thinsp;10%) above the reference sequences. Results were obtained for the full set of 11 core VNTR loci covered by Deeplex Myc-Lep for this sample. VNTR markers are presented in the following order: 6-3a, AC8a, AC8b, AC9, GTA9, GAA21, GGT5, 6\u0026ndash;7, 12\u0026thinsp;\u0026minus;\u0026thinsp;5, 21\u0026thinsp;\u0026minus;\u0026thinsp;3, and 23\u0026thinsp;\u0026minus;\u0026thinsp;3. Abbreviations: RIF, rifampicin; DPS, dapsone; FQ, fluoroquinolones; VNTR, variable-number tandem repeat; SNP, single nucleotide polymorphism.\u003c/p\u003e\n \u003cp\u003eBased on multi-locus VNTR patterns, a dendrogram was constructed to assess genetic similarity among samples. This analysis identified three distinct genetic patterns, referred to as Groups A, B, and C (Supplementary Fig. 1B). These groups were distinguished by specific allele combinations, particularly at GAA21, which showed clear differentiation: higher allele values in Group A, intermediate in Group B, and lower in Group C (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRepeat polymorphisms of 9 polymorphic VNTR loci with the Hunter-Gaston discriminatory index (H) reported for each locus.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eVillage\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eGAA21\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eGTA9\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eAC8a\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003eAC9\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e6-3a\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003eAC8b\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003eGGT5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e12\u0026thinsp;\u0026minus;\u0026thinsp;5\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e23\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c12\"\u003e\n \u003cp\u003eSNP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c13\"\u003e\n \u003cp\u003eSubtype\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c14\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eMarotongo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c12\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c13\"\u003e\n \u003cp\u003eD-Malagasy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c14\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAnkazondrigintra\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c12\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c13\"\u003e\n \u003cp\u003eD-Malagasy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c14\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAmbalakida\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c12\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c13\"\u003e\n \u003cp\u003eD-Malagasy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c14\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAndrafiabe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c12\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c13\"\u003e\n \u003cp\u003eD-Malagasy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c14\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAndrafiabe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c12\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c13\"\u003e\n \u003cp\u003eD-Malagasy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c14\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9068\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eAntsoha\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c12\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c13\"\u003e\n \u003cp\u003eD-Malagasy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c14\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003e9048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eBesakoa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c12\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c13\"\u003e\n \u003cp\u003eD-Malagasy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c14\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c8\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c9\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c10\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c11\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eID: patient identifier; Village: village of origin of the patients within the Miandrivazo district; VNTR: Variable Number Tandem Repeat (6-3a, AC9, AC8b, GAA21, GGT5, AC8a, GTA9, 12\u0026thinsp;\u0026minus;\u0026thinsp;5, 23\u0026thinsp;\u0026minus;\u0026thinsp;3), H: Hunter-Gaston discriminatory index; SNP: Single Nucleotide Polymorphism. Group: cluster defined by VNTR analysis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe PEOPLE study, conducted in Miandrivazo district, identified 80 new leprosy cases among 20,221 individuals who were clinically examined through door-to-door screening over a three-year period. 70 were detected at baseline in 2019, corresponding to a prevalence of 34/10,000 inhabitants, while the remaining cases were identified in 2020 and 2021, with an incidence of 4.95/10,000 inhabitants[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Among them, 75 were undergoing MDT treatment at the time of sampling, while five had not yet been treated, 4 of whom underwent biological sampling. Delays in sample collection were mainly due to COVID-19 related lockdowns, which prevented teams from Antananarivo from traveling to Miandrivazo. Also, even when local staff identified cases, on-site sampling before onset of treatment was not feasible due to the lack of test consumables.\u003c/p\u003e \u003cp\u003eAmong the 12 molecularly analysable biopsy samples, representing 15% of the cohort, no mutations associated with resistance to rifampicin, dapsone, or fluoroquinolones were detected by any of the three different assays, based either on separate or combined results. These findings indicate that the current treatment regimen in the region remains effective. Furthermore, the sensitivity of circulating strains to rifampicin supports the potential effectiveness of its use as post-exposure prophylaxis among contacts during the PEOPLE study. Our results are similar to those reported in the Comoros, a neighboring country: most strains had the same 1D genotype (supposedly of Malagasy subtype), and \u003cem\u003eM. leprae\u003c/em\u003e remained fully sensitive to rifampicin, fluoroquinolones, and dapsone in 216 patients [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the limited sample size analyzed in this pilot study warrants caution in interpreting these results, as they may underestimate the true prevalence of drug resistance. Increasing the number of tested samples would strengthen surveillance, especially since rifampicin resistance has already been observed for other diseases in Madagascar, such as tuberculosis (1.3% of patients between 2012 and 2017) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Globally, resistance to anti-leprosy drugs remains a concern. A meta-analysis of 4,454 patients reported resistance rates of 5.1% for rifampicin, 7.4% for dapsone, 1.8% for clofazimine, and 3.5% for ofloxacin, with regional variations (12.5% in Africa, 7.5% in the Americas, 16.9% in Europe)[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Despite possible bias due to excluded studies, these findings highlight the need for continuous surveillance to detect emerging resistance and maintain MDT effectiveness.\u003c/p\u003e \u003cp\u003eIn addition to resistance monitoring, molecular characterization of circulating strains provided complementary insights into local transmission dynamics. Strain genotyping showed homogeneity with the exclusive presence of the genotype 1D-Malagasy, consistent with the dominance of this genotype in other Madagascar settings reported by Avanzi \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This homogeneity may reflect a local and persistent circulation of these strains. However, the detection of genotype 1A in two patients from the Boeny region, as reported by \u003cem\u003eAvanzi et al\u003c/em\u003e. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], suggests that this strain likely also circulates in other regions, potentially including the Menabe areas studied here. Larger studies are needed to complement these existing observations, confirm the distribution of genotypes, and better understand the persistence and transmission of \u003cem\u003eM. leprae\u003c/em\u003e strains in Madagascar.\u003c/p\u003e \u003cp\u003eDespite the small size of this pilot dataset, VNTR analysis remarkably differentiated all analysable strains sharing the same SNP 1D-Malagasy genotype, with GAA21, GTA9, and AC8a highlighted as highly discriminatory markers. The epidemiological significance of these unique genotypic profiles is supported by the fact that they originated from different neighborhoods and households, with no known contacts between them. These initial observations are consistent with the well-recognized high-resolution power of VNTR typing, making it particularly suited to fine-scale studies of \u003cem\u003eM. leprae\u003c/em\u003e transmission over short epidemiological distances. As doable by utilizing the Deeplex Myc-Lep assay, combining SNP and VNTR approaches will therefore be valuable to identify distinct regional clusters within the Malagasy SNP 1D genotype, thereby improving the understanding of \u003cem\u003eM. leprae\u003c/em\u003e transmission dynamics and spatial structuring in Madagascar.\u003c/p\u003e \u003cp\u003eThe RLEP qPCR test proved essential as a triage tool before resistance analysis (LiPA, Sanger, and Deeplex Myc-Lep). A Ct threshold for positivity of \u0026lt;\u0026thinsp;27 was set to maximize chances of successful results across the three tests, as sensitivity declines beyond this point. In our cohort, which was predominantly composed of patients under treatment, the detection rate of \u003cem\u003eM. leprae\u003c/em\u003e by RLEP qPCR from SB was 85%, suggesting that the clinical leprosy diagnosis was overall highly accurate. The result is comparable to those reported in untreated patients by Martinez \u003cem\u003eet al\u003c/em\u003e. (87%)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and Azevedo et al. (84.9%) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. RLEP qPCR was highly sensitive for detecting and quantifying \u003cem\u003eM. leprae\u003c/em\u003e DNA in both PB and MB cases. However, it does not allow distinction between viable and non-viable bacilli [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Complementary approaches, such as mouse footpad inoculation, RNA-based viability assays [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], or the morphological index on microscopy, would therefore have been necessary, which, unfortunately, was not feasible in this study.\u003c/p\u003e \u003cp\u003eYet, even after several months of treatment, RLEP qPCR remains sufficiently sensitive to detect and quantify \u003cem\u003eM. leprae\u003c/em\u003e DNA in skin lesions. One patient was RLEP qPCR positive (Ct\u0026thinsp;=\u0026thinsp;16.6) after six months of treatment, based on both SB and NS samples. This patient also was seropositive for anti-PGL-I IgM. However, these results do not allow conclusions regarding bacterial viability or the patient\u0026rsquo;s infectiousness, as these markers may reflect the presence of non-viable bacilli or bacterial debris. It has been widely reported that IgM levels against \u003cem\u003eM. leprae\u003c/em\u003e PGL-I strongly correlate with bacterial load in both humans and animals[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, these antibodies are reduced upon efficient treatment of patients.[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, anti-PGL-I antibodies do not confer protection against leprosy [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Due to the fact that most patients in this study were sampled only during treatment and not at diagnosis, no statements can be made here other than the PGL-I rapid test using FSB was applicable in the field and that the anti-PGL-I IgM levels inversely correlated with Ct values as previously found [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, the elevated acceptance of NS and FSB samples suggests that these low-invasive methods are well tolerated by leprosy patients in Madagascar. Their simplicity, already widely used for other diseases in the country, contributes to higher acceptance compared to SB, which are slightly uncomfortable and require local anesthetics. These types of samples have been employed in several countries for the detection of \u003cem\u003eM. leprae\u003c/em\u003e-infected individuals as well as to identify active cases. FSB samples can enable the detection of immunological biomarkers related to infection and disease, including antibodies, chemokines, cytokines and acute phase proteins. When quantified, the levels of these host biomarkers allow differentiation of various clinical phenotypes of leprosy (MB and PB) from control individuals [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In this context, a study is currently underway to validate several of these host biomarkers in patients from Madagascar.\u003c/p\u003e \u003cp\u003eA major limitation of our study is the relatively small number of samples analyzed, with most patients already undergoing anti-leprosy treatment at the time of sampling and the absence of AFB smear results.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe results from this pilot study suggest that \u003cem\u003eM. leprae\u003c/em\u003e strains circulating in the Miandrivazo district in Madagascar are sensitive to the main anti-leprosy drugs. RLEP qPCR was implemented in the reference laboratory to pre-select samples with sufficient DNA levels, defined by a Ct threshold of \u0026lt;\u0026thinsp;27, which ensured successful results of the three molecular drug-resistance assays utilized. The 1D-Malagasy genotype remains predominant in this district. However, VNTR analysis detected all analyzable strains of this type, suggesting the possibility of extending this approach at a larger regional or national scale to obtain better insights into the circulation and transmission of individual strains.\u003c/p\u003e \u003cp\u003eDespite the limited size of this pilot study, these findings provide a solid basis for strengthening drug resistance surveillance and genotyping of \u003cem\u003eM. leprae\u003c/em\u003e strains in Madagascar, offering perspectives for more effective leprosy control in the country.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eWHO (World Health Organization), MB (Multibacillary); PB (Paucibacillary); MDT (Multi-drug therapy); PEP (Post-Exposure Prophylaxis); FSB (Finger stick blood); NS (Nasal swab)\u003c/p\u003e\n\u003cp\u003eSB (Skin biopsy); Ct (Cycle threshold); R (Ratio); UCP-LF (up-converting \u0026nbsp;reporter particles Lateral Flow); (PGL-I – Phenolic Glycolipid-I); IgM (Immunoglobulin M); \u003cem\u003eM. leprae (Mycobacterium leprae);\u003c/em\u003e qPCR (Quantitative polymerase chain reaction); RLEP (Repetitive element of \u003cem\u003eM. leprae\u0026nbsp;\u003c/em\u003egenome); SNP (Single nucleotide polymorphism); VNTR (Variable number tandem repeat); H (Hunter Gaston diversity index); CERBM (Comité d’Éthique de la Recherche Biomédicale); PEOPLE (Post-Exposure Prophylaxis for Leprosy); LiPA (Line Probe Assay); gyrA ( Gene encoding DNA gyrase subunit A); gyrB ( Gene encoding DNA gyrase subunit B); folP1 ( Gene encoding Dihydropteroate synthase); rpoB – Gene encoding RNA polymerase β subunit).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki (7th revision, 64th meeting, 2013, Fortaleza). Ethical approval was obtained from the “Comité d’Éthique de la Recherche Biomédicale” (CERBM) in Madagascar. The trial was registered in the public ClinicalTrials.gov registry (NCT03662022, September 7, 2018, https://clinicaltrials.gov/ct2/show/NCT03662022). Written informed consent was obtained from each participant or, for participants under 18 years of age, from a parent or guardian. Additional consent was obtained from leprosy patients for biological sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is part of the PEOPLE project, which is embedded in the EDCTP2 programme and funded by the European Union (grant number RIA2017NIM-1847-PEOPLE). The views and opinions expressed in this publication are solely those of the authors and do not necessarily reflect those of EDCTP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: BCD, EC, BC, MA, PC, EH, AG.\u003c/p\u003e\n\u003cp\u003eData curation: LM, SR, TR, AJ, PS.\u003c/p\u003e\n\u003cp\u003eFormal analysis: LM, SR, BC, JLB, SB, AJ, PS, AG.\u003c/p\u003e\n\u003cp\u003eFunding acquisition: BCD, EC, MA, EH, LHS, BC, AG.\u003c/p\u003e\n\u003cp\u003eMethodology: \u0026nbsp;SR, LM, SMB, DJ, PS, BCD, EC, AG.\u003c/p\u003e\n\u003cp\u003eSample collection: SR, LM, LMA, AR, BC.\u003c/p\u003e\n\u003cp\u003eSupervision: BC, SR, SMB, TM, EC, BCD, AG.\u003c/p\u003e\n\u003cp\u003eVisualization: LM, JLB, AG.\u003c/p\u003e\n\u003cp\u003eWriting original draft: LM, SR, JLB, TR, AG.\u003c/p\u003e\n\u003cp\u003eWriting review and editing: LM, SR, SMB, PC, DJ, AR, LMA, MA, JLB, EC, AJ, PS, EH, BCD, BC, TR, LHS, AG.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our sincere gratitude to the medical teams on the ground for their dedication to the door-to-door active screening and sample collection. We also thank the teams at NLP Madagascar and the Raoul Follereau Foundation for their support. Finally, we extend our sincere appreciation to all participants and patients who contributed to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' information (optional)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003eCentre d’Infectiologie Charles Mérieux, Madagascar (CICM), \u003cstrong\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003eDept of Leiden University Center of Infectious Diseases (LUCID), Leiden University Medical Center, Leiden, the Netherlands, \u003cstrong\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/strong\u003eRaoul Follereau Foundation (FRF), Antananarivo, Madagascar, \u003cstrong\u003e\u003csup\u003e4\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003eInstitute of Tropical Medicine (IMT), Antwerp, Belgium,\u003csup\u003e5\u003c/sup\u003eDept. of Cel and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands, \u003cstrong\u003e\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003eNational Leprosy Program (NLP), Madagascar, \u003cstrong\u003e\u003csup\u003e7\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003eFondation Mérieux Lyon, \u003csup\u003e8\u003c/sup\u003e INSERM, IAME UMR1137, Université de Paris Cité 75018 Paris, France,\u003csup\u003e\u0026nbsp;\u003cstrong\u003e9\u003c/strong\u003e\u003c/sup\u003eUniv. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019–UMR 9017, Center for Infection and Immunity of Lille, Lille, France.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGlobal leprosy update, 2018: moving towards a leprosy-free world [Internet]. [cit\u0026eacute; 12 janv 2026]. Disponible sur: https://www.who.int/publications/i/item/who-wer9435-36\u003c/li\u003e\n \u003cli\u003eCambau E, Saunderson P, Matsuoka M, Cole ST, Kai M, Suffys P, et al. Antimicrobial resistance in leprosy: results of the first prospective open survey conducted by a WHO surveillance network for the period 2009\u0026ndash;15. Clin Microbiol Infect. 2018;24:1305‑10. doi:10.1016/j.cmi.2018.02.022 PubMed PMID: 29496597; PubMed Central PMCID: PMC6286419.\u003c/li\u003e\n \u003cli\u003eGlobal leprosy (\u0026lrm;Hansen disease)\u0026lrm; update, 2023: Elimination of leprosy disease is possible \u0026ndash; Time to act! [Internet]. [cit\u0026eacute; 12 janv 2026]. 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Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-Lep. eBioMedicine. 2023;93. doi:10.1016/j.ebiom.2023.104649 PubMed PMID: 37327675.\u003c/li\u003e\n \u003cli\u003eBraet SM, Jouet A, Aubry A, Dyck-Lippens MV, Lenoir E, Assoumani Y, et al. Investigating drug resistance of Mycobacterium leprae in the Comoros: an observational deep-sequencing study. Lancet Microbe. 2022;3:e693‑700. doi:10.1016/S2666-5247(22)00117-3 PubMed PMID: 35850123.\u003c/li\u003e\n \u003cli\u003ePierneef L, Braet SM, Jong D de, Ramboarina S, Hooij A van, Abdou MW, et al. Large-scale field application of a fingerstick blood test for Mycobacterium leprae infection: Monitoring population-wide effects of case finding and post-exposure prophylaxis on transmission in the Comoros and Madagascar. PLOS Glob Public Health. 2025;5:e0005270. doi:10.1371/journal.pgph.0005270\u003c/li\u003e\n \u003cli\u003eGeluk A, van Meijgaarden KE, Wilson L, Bobosha K, van der Ploeg-van Schip JJ, van den Eeden SJF, et al. Longitudinal immune responses and gene expression profiles in type 1 leprosy reactions. J Clin Immunol. 2014;34:245‑55. doi:10.1007/s10875-013-9979-x PubMed PMID: 24370984.\u003c/li\u003e\n \u003cli\u003eKhadge S, Banu S, Bobosha K, van der Ploeg-van Schip JJ, Goulart IM, Thapa P, et al. Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal. BMC Infect Dis. 2015;15:477. doi:10.1186/s12879-015-1128-0 PubMed PMID: 26510990; PubMed Central PMCID: PMC4625471.\u003c/li\u003e\n \u003cli\u003eTi\u0026oacute;-Coma M, van Hooij A, Bobosha K, van der Ploeg-van Schip JJ, Banu S, Khadge S, et al. Whole blood RNA signatures in leprosy patients identify reversal reactions before clinical onset: a prospective, multicenter study. Sci Rep. 2019;9:17931. doi:10.1038/s41598-019-54213-y PubMed PMID: 31784594; PubMed Central PMCID: PMC6884598.\u003c/li\u003e\n \u003cli\u003eGuidelines for the diagnosis, treatment and prevention of leprosy [Internet]. [cit\u0026eacute; 31 oct 2025]. Disponible sur: https://www.who.int/publications/i/item/9789290226383\u003c/li\u003e\n \u003cli\u003eCorstjens PLAM, de Dood CJ, van der Ploeg-van Schip JJ, Wiesmeijer KC, Riuttam\u0026auml;ki T, van Meijgaarden KE, et al. Lateral flow assay for simultaneous detection of cellular- and humoral immune responses. Clin Biochem. 2011;44:1241‑6. doi:10.1016/j.clinbiochem.2011.06.983 PubMed PMID: 21763300; PubMed Central PMCID: PMC3177995.\u003c/li\u003e\n \u003cli\u003eWoods SA, Cole ST. A rapid method for the detection of potentially viable Mycobacterium leprae in human biopsies: a novel application of PCR. FEMS Microbiol Lett. 1989;53:305‑9. doi:10.1016/0378-1097(89)90235-8 PubMed PMID: 2693204.\u003c/li\u003e\n \u003cli\u003eBraet SM, Hooij A van, Hasker E, Fransen E, Wirdane A, Baco A, et al. Minimally invasive sampling to identify leprosy patients with a high bacterial burden in the Union of the Comoros. PLoS Negl Trop Dis. 2021;15:e0009924. doi:10.1371/journal.pntd.0009924\u003c/li\u003e\n \u003cli\u003ePierneef L, Malaviya P, van Hooij A, Sundar S, Singh AK, Kumar R, et al. Field-friendly anti-PGL-I serosurvey in children to monitor Mycobacterium leprae transmission in Bihar, India. Front Med. 2023;10:1260375. doi:10.3389/fmed.2023.1260375 PubMed PMID: 37828950; PubMed Central PMCID: PMC10565223.\u003c/li\u003e\n \u003cli\u003ePierneef L, van Hooij A, de Jong D, Wassenaar G, Verhard E, Tjon Kon Fat E, et al. Rapid test for Mycobacterium leprae infection: a practical tool for leprosy. Infect Dis Poverty. 2024;13:88. doi:10.1186/s40249-024-01262-9\u003c/li\u003e\n \u003cli\u003eA guide for surveillance of antimicrobial resistance in leprosy: 2017 update [Internet]. [cit\u0026eacute; 12 janv 2026]. Disponible sur: https://www.who.int/publications/i/item/9789290225492\u003c/li\u003e\n \u003cli\u003eCollins JH, Lenz SM, Ray NA, Lahiri R, Adams LB. Assessment of esxA, hsp18, and 16S transcript expression as a measure of Mycobacterium leprae viability: A comparison with the mouse footpad assay. Lepr Rev. 2023;94:7‑18. doi:10.47276/lr.94.1.7\u003c/li\u003e\n \u003cli\u003eTurankar RP, Singh V, Lavania M, Singh I, Sengupta U, Jadhav RS. Existence of viable Mycobacterium leprae in natural environment and its genetic profiling in a leprosy endemic region. Front Trop Dis. 2022;3. doi:10.3389/fitd.2022.972682\u003c/li\u003e\n \u003cli\u003eZivarifar H, Ahrari F, Karbalaei M. 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Front Microbiol. 2024;15. doi:10.3389/fmicb.2024.1497319\u003c/li\u003e\n \u003cli\u003evan Hooij A, Tjon Kon Fat EM, van den Eeden SJF, Wilson L, Batista da Silva M, Salgado CG, et al. Field-friendly serological tests for determination of M. leprae-specific antibodies. Sci Rep. 2017;7:8868. doi:10.1038/s41598-017-07803-7 PubMed PMID: 28827673; PubMed Central PMCID: PMC5566372.\u003c/li\u003e\n \u003cli\u003eSchilling AK, van Hooij A, Corstjens P, Lurz PWW, DelPozo J, Stevenson K, et al. Detection of humoral immunity to mycobacteria causing leprosy in Eurasian red squirrels (Sciurus vulgaris) using a quantitative rapid test. Eur J Wildl Res. 2019;65:49. doi:10.1007/s10344-019-1287-1\u003c/li\u003e\n \u003cli\u003eRoche PW, Britton WJ, Failbus SS, Neupane KD, Theuvenet WJ. Serological monitoring of the response to chemotherapy in leprosy patients. Int J Lepr Mycobact Dis Off Organ Int Lepr Assoc. 1993;61:35‑43. PubMed PMID: 8326179.\u003c/li\u003e\n \u003cli\u003eKhadge S, Banu S, Bobosha K, van der Ploeg-van Schip JJ, Goulart IM, Thapa P, et al. Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal. BMC Infect Dis. 2015;15:477. doi:10.1186/s12879-015-1128-0 PubMed PMID: 26510990; PubMed Central PMCID: PMC4625471.\u003c/li\u003e\n \u003cli\u003eHooij A van, Meijgaarden KE van, Khatun M, Soren S, Walburg K, Alam K, et al. Integrative immune analysis in patients with leprosy reveals host factors associated with mycobacterial control. eBioMedicine. 2025;118. doi:10.1016/j.ebiom.2025.105855 PubMed PMID: 40683203.\u003c/li\u003e\n \u003cli\u003evan Hooij A, Tjon Kon Fat EM, de Jong D, Khatun M, Soren S, Chowdhury AS, et al. Prototype multi-biomarker test for point-of-care leprosy diagnostics. iScience. 2020;24:102006. doi:10.1016/j.isci.2020.102006 PubMed PMID: 33490914; PubMed Central PMCID: PMC7807156.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycobacteria","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Mycobacteria](https://mycobacteria.biomedcentral.com/)","snPcode":"44350","submissionUrl":"https://submission.springernature.com/new-submission/44350/3","title":"Mycobacteria","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mycobacterium leprae, Madagascar, RLEP qPCR, Drug resistance, Genotype, 1D-Malagasy, anti-PGL-I serology","lastPublishedDoi":"10.21203/rs.3.rs-9335540/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9335540/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eTo curb the emergence of antibiotic-resistant \u003cem\u003eMycobacterium leprae\u003c/em\u003e strains, the WHO recommends monitoring of antimicrobial resistance (AMR). In Madagascar, molecular surveillance of resistance to anti-\u003cem\u003eM. leprae\u003c/em\u003e drugs, remains limited geographically and in terms of quantitative data. A clinical trial on leprosy prophylaxis (PEOPLE) conducted in the district of Miandrivazo provided an opportunity to explore the feasibility of an integrated approach for improving leprosy surveillance and resistance monitoring in Madagascar. This study aimed to i) determine the genetic diversity of the identified \u003cem\u003eM. leprae\u003c/em\u003e strains, (ii) detect mutations associated with AMR, (iii) evaluate and compare the acceptance rate of low-invasive sampling methods with that of skin biopsies, and (iv) assess seropositivity for anti-\u003cem\u003eM. leprae\u003c/em\u003e PGL-I IgM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003ePatients were identified through door-to-door screening and skin biopsies (SB), fingerstick blood (FSB) and nasal swabs (NS) were collected. SB were analyzed by RLEP qPCR for detection of \u003cem\u003eM. leprae\u003c/em\u003e DNA, resistance testing using LiPA (Line Probe Assay), and resistance analysis and SNP and/or VNTR genotyping using Sanger sequencing and Deeplex Myc-Lep. NS and FSB samples were used to detect bacterial colonization by RLEP qPCR and detect past and/or present infection by measuring anti-\u003cem\u003eM. leprae\u003c/em\u003e PGL-I IgM levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e80 leprosy patients were identified, including 41 multibacillary and 39 paucibacillary. Out of these, 57 accepted NS, 47 FSB, and 33 SB. High levels of anti-PGL-I IgM were detected in 6/47 FSB samples. RLEP qPCR was positive in 4/57 NS samples and 28/33 SB samples. Among SB positive, 12 yielded interpretable results when combining data from LiPA, Deeplex Myc-Lep, and Sanger sequencing. No drug resistance conferring mutations were detected. All strains belonged to the 1D-Malagasy genotype, and VNTR analysis for 7 samples with results available across 9 loci showed 7 unique types, with loci GAA21, GTA9, and AC8a were the most informative for strain differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eDespite the small sample size, this study shows that NS and FSB samples are well accepted in patients. No mutations conferring resistance were identified and the \u003cem\u003eM. leprae\u003c/em\u003e strains belong to the 1D-Malagasy genotype. These findings support the feasibility of field-based molecular surveillance and provide a foundation for expanding AMR monitoring in Madagascar.\u003c/p\u003e","manuscriptTitle":"Assessing improved surveillance of leprosy drug resistance in Madagascar: A pilot study in the Miandrivazo district","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 15:10:56","doi":"10.21203/rs.3.rs-9335540/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-29T10:24:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T14:25:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123189311003039319810750446280044190326","date":"2026-04-16T16:48:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47431061344405291572565947248778383968","date":"2026-04-14T04:11:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271219800332128162797157061381462165850","date":"2026-04-14T04:09:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-13T21:47:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T01:59:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T01:58:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycobacteria","date":"2026-04-06T15:36:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycobacteria","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Mycobacteria](https://mycobacteria.biomedcentral.com/)","snPcode":"44350","submissionUrl":"https://submission.springernature.com/new-submission/44350/3","title":"Mycobacteria","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69bffcb3-7825-416d-aaa2-a8f74d680180","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-04-29T10:24:44+00:00","index":17,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T15:10:56+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 15:10:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9335540","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9335540","identity":"rs-9335540","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}