BIOMEDICAL REPORTS 15: 88, 2021

A 5‑year evaluation of chemoprophylactic treatment in elementary school children with subclinical leprosy

LINDA ASTARI1‑4, NASTITI INTAN PERMATA SARI5, KUSNARTEDJO NANANG6, EDY CAHYONO6, NANNY HERWANTO2,3, MUHAMMAD YULIANTO LISTIAWAN2,3, DINAR ADRIATY4, ISWAHYUDI4, RATNA WAHYUNI4,7, MEDHI DENISA ALINDA2,3, BAGUS HARYO KUSUMAPUTRA2,3, REGITA INDIRA AGUSNI2,3, INDROPO AGUSNI2‑4, SHINZO IZUMI4 and CITA ROSITA SIGIT PRAKOESWA2‑4

1Doctoral Program of Medical Science; 2Department of Dermatovenereology, Faculty of Medicine, Universitas Airlangga, , 60131; 3Dr. General Academic Hospital, Surabaya, East Java 60285; 4Institute of Tropical Disease, Universitas Airlangga, Surabaya, East Java 60115; 5Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Pertahanan, , 16810; 6Sumberglagah Leprosy Hospital, Mojokerto, East Java 67152; 7Department of Health, Faculty of Vocational Studies, Universitas Airlangga, Surabaya East Java 60286, Indonesia

Received March 16, 2020; Accepted June 2, 2021

DOI: 10.3892/br.2021.1464

Abstract. Subclinical leprosy is an infectious disease 200 were seropositive. The anti‑PGL‑1 IgM antibody levels in which the immune system remains infected with significantly decreased between 2011 and 2015 in Nguling (from Mycobacterium leprae (M. leprae). The progress of subclin‑ 1,066.7 to 137.4 U/ml) and Raas (from 773.1 to 563.4 U/ml), ical leprosy to clinical cases within 1 year of infection is 1.5%, the levels decreased every year. In addition, the proportion of with an increase to 6% in the following 4 years. Rifampicin is patients with decreased anti‑PGL‑1 IgM antibody levels was frequently used for prevention of leprosy, and clarithromycin consistently higher than patients with increased anti‑PGL‑1 has a bactericidal effect on M. leprae. Thus, the combination IgM antibody levels in all periods, except during 2013‑2014, of both is expected to improve disease control in patients with in Nguling and Raas. Chemoprophylactic treatment involving subclinical leprosy. The aim of the present study was to eval‑ rifampicin and clarithromycin may thus be effective against uate the efficacy of a chemoprophylactic treatment involving subclinical leprosy amongst elementary school children. rifampicin and clarithromycin against subclinical leprosy in elementary school children from endemic areas of East Java Introduction over a 5‑year period. The study was performed between 2011 and 2015. Samples were collected from 2,548 healthy In 2015, Indonesia was amongst the three countries (along elementary school children in Nguling (Pasuruan) and Raas with India and Brazil) which reported >10,000 new leprosy (Sumenep), and analysed using ELISA for anti‑PGL (phenolic cases, contributing to 8% of new cases globally. Amongst glycolipid)‑1 IgM antibodies. Children who were seropositive all new patients with leprosy in Indonesia, 10% had already for anti‑PGL‑1 IgM antibodies received a chemoprophylactic progressed to disability on diagnosis, and 11% were chil‑ regimen consisting of rifampicin (300 mg/day) and clarithro‑ dren (1). Leprosy in children is quite common in endemic mycin (250 mg/day) daily for the initial 10 days, followed by countries. Moreover, 20‑30% of patients with new cases the same regimen every 2 weeks for 3 months. Clinical and exhibit neuritis and some are diagnosed with disability (2). serological evaluations were performed annually for 5 years. The younger patients (<15 years) show active circulation and Amongst the 2,548 healthy elementary school children, transmission of Mycobacterium leprae (M. leprae) within the community, thus reflecting inefficiency in the national health system (3‑5). With regard to leprosy in children, the most important matter is the prevention of disability and deformi‑ ties by early detection and improved treatment of leprosy in Correspondence to: Professor Cita Rosita Sigit Prakoeswa, leprosy endemic areas (6). Leprosy in children, particularly Department of Dermatovenereology, Faculty of Medicine, Universitas Airlangga, Jl. Prof. Dr. no. 47, Surabaya, East Java 60131, subclinical leprosy, may occur as currently, treatments are Indonesia focused only on clinical leprosy, and subclinical leprosy is E‑mail: cita‑[email protected] not a focus, even though patients with subclinical leprosy are sources of further infection. Key words: Mycobacterium leprae, subclinical leprosy, clinical A healthy individual exhibiting a sufficient immune response leprosy, clarithromycin, infectious disease will typically attack an M. leprae infection; however, an indi‑ vidual with a compromised immune system may not be able to 2 ASTARI et al: EVALUATION OF A CHEMOPROPHYLACTIC REGIMEN IN CHILDREN WITH SUBCLINICAL LEPROSY fend of the M. leprae infection appropriately, and this condition children from endemic areas in East Java (Indonesia) over a is considered subclinical leprosy (7). Subclinical leprosy can 5‑year period. develop into clinical leprosy within 2‑10 years, depending on the individual's immune status (8,9). In 2008, the International Materials and methods Leprosy Congress (Hyderabad) showed that 1.5% of subclinical leprosy cases progress to clinical leprosy cases within 1 year, Criteria. The primary criteria for the present study were which increases to 6% in the following 4 years (5,10,11). Thus, as follows: High endemicity (≥250 new cases per year in it is crucial to prevent the progression of subclinical leprosy to the last 3 years), epidemiological data available for at clinical leprosy, as the latter is more infectious and potentially least the last 10 years, population not highly mobile, good more debilitating for the patient (5,10,11). Using sensitive and collaboration with government health services, willingness specific screening tests is important to control transmission to integrate prophylactic treatment involving leprosy health of leprosy. Serological tests have become the most common services, high logistical feasibility and good health infra‑ approach to detect subclinical infection (5,10,11). PGL‑1, structure (including adequate leprosy services and contact a specific M. leprae cell wall antigen, is a popular antigen screening). assessed for using antibodies, as its presence can be detected in the serum. Detection using these antibodies can be used for Patients. The present study was performed annually between diagnosis, prediction of recurrence and identification of the 2011 and 2015. Samples were collected from 2,548 healthy risk of spreading the disease amongst individuals or within elementary school students who were not contacts of patients the community (5,10,11). A meta‑analysis by Penna et al (12) with leprosy. The students were recruited from several concluded that the risk of spreading from patients with clinical elementary schools in one area, but the school grades differed leprosy is ~3X higher amongst patients positive for anti‑PGL‑1 according to age. All students obtained consent from their antibodies than those who were negative for these antibodies. parents to participate in the present study, and signed an Furthermore, it has been shown that leprosy contacts (13,14) informed consent form that clearly stated the purpose of the and population (15) in an leprosy endemic area, with IgM research, the method of sample collection, and the lack of anti‑PGL‑1 seropositivity have a 6‑fold higher relative risk of risk from medical procedures. They also received an agree‑ becoming ill from leprosy in the 6‑10 years of follow‑up. ment form and resignation form. The present study was Various types of chemoprophylactic approaches have approved by the Ethical Committee in Health Research Unit of shown favourable results for prevention of leprosy infection or Dr. Soetomo General Hospital Surabaya, Indonesia (approval subclinical leprosy. In a previous study, intermittent dapsone no. 30‑511/H3.13/PPd/2012). administration for 2 years and intermittent acedapsone admin‑ The recruited cohort consisted of 39 males and 59 females, istration for 7 months reduced the risk of leprosy by 60 and 49%, and the age ranged from 7‑14 years old. The regions from which respectively (16). The single‑dose rifampicin (SDR) chemopro‑ patients were recruited were located in East Java, Indonesia; phylactic regimen is currently being used in Indonesia, as well specifically, Nguling (Pasuruan) and Raas (Sumenep). as in India, as well as several other ASEAN countries (17). Collection of samples in this study was performed by home However, this regimen has been shown to reduce the incidence visits during the 5 years of observation, and was completed of leprosy by only 57% (18). The efficacy of this regimen is using portable equipment. Peripheral blood samples were more distinguished in distant contacts than in close contacts of collected from one finger following disinfection. The collec‑ index patients and may be associated with the bacillary load, tion of the blood used a capillary pipette; 100 µl was collected, which is supposedly higher in close contacts than in infrequent and this was then placed on a filter paper (Whatman, PLC; contacts (19,20). Furthermore, studies have shown that SDR GE Healthcare Life Sciences; cat. no. 1442 110) until dry to is effective for the prevention of leprosy only in those who avoid contaminants, such as fungi (26). receive a regimen; as such, SDR should be ideally administered to entire communities for it to exert an effect at the commu‑ Serological test. The samples were analysed using indirect nity level (19,21). Therefore, early detection of index cases, ELISA using anti‑PGL‑1 (NTP‑BSA; cat. no. Nara XVII‑48) increased coverage of SDR post‑exposure prophylaxis (PEP), according to the manufacturer's protocol for anti‑PGL‑1 detection of subclinical leprosy followed by direct PEP and IgM antibodies (27). A level of 605 U/ml or above was enhancement of the PEP regimen (PEP++ regimen) are required considered to indicate seropositivity. Antibody titres were clas‑ to improve prevention programs. The improved regimen is sified as follows: +1, 605‑1,000 U/ml; +2, 1,001‑2,000 U/ml; expected to be sufficiently effective for the treatment of patients +3, 2,001‑3,000 U/ml; and +4, >3,000 U/ml. with subclinical leprosy who possess a high bacillary load. Clarithromycin exhibits a bactericidal effect on M. leprae Treatment regimens. Participants who were seropositive for and has an important role in the treatment of rifampicin‑resis‑ anti‑PGL‑1 IgM antibodies received a chemoprophylactic tant leprosy (22‑24). The pharmacokinetics are promising regimen involving rifampicin (300 mg/day) and clarithromycin given its ability to penetrate well into macrophages and leuko‑ (250 mg/day) every day for the initial 10 days and then received cytes (4,25), which are the targets of M. leprae (5). Thus, a the same regimen every 2 weeks for 3 months. Evaluation of combination of clarithromycin and rifampicin may facilitate clinical and serological positivity was performed every year improved disease control in patients with subclinical leprosy. for 5 years (28,29). The aim of the present study was to evaluate the efficacy of a chemoprophylactic treatment involving rifampicin and clar‑ Data presentation. Descriptive analysis was used for the ithromycin against subclinical leprosy in elementary school results of the present study. Data presentation was performed BIOMEDICAL REPORTS 15: 88, 2021 3

Table I. Mean of anti‑PGL‑1 IgM antibody levels in the chil‑ the participants. The samples can be dried and stored for a dren recruited from Nguling and Raas between 2011 and 2015. relatively long time prior to analysis or referral (21). Therefore, this procedure may be suitable to support the sustainability Mean of early detection and prophylactic programmes. However, a ------previous study showed that anti‑PGL‑1 antibody was not an Year Nguling Raas efficient predictor of contacts that may develop leprosy in endemic areas, and suggested that more specific biomarkers 2011 1066,71875 773,123077 are required for early detection of leprosy (30). Another study 2012 685,15625 560,923077 showed that the sensitivity of anti‑PGL‑1 antibody for early 2013 450,59375 281,276923 detection of clinical leprosy was <50%, although the speci‑ 2014 599 411,353846 ficity was 80% (3). Nevertheless, as mentioned in a study by 2015 137,4375 5,634 Barreto et al (3), several reports have found higher levels of anti‑PGL‑1 antibodies in children and young adults compared with older adults (3). Moreover, this previous study reported higher levels of anti‑PGL‑1 antibodies in children living in using Microsoft Excel version 16.42 (Microsoft Corporation), rural areas (considered to be busy areas facing food shortages) and included frequency (raw data) and mean of the anti‑PGL‑1 than in children living in high‑income areas. levels (Table I). Previous studies on leprosy contacts (13,14) or on blood donors representing the population of an endemic area in Results south eastern Brazil (15) found a 6‑fold higher relative risk of illness in individuals who were IgM anti‑PGL‑1 seropositive. Among the 2,548 healthy elementary school children, The present study showed a promising result of a decrease 200 were seropositive (79 from Nguling and 121 from Raas). in the average levels of anti‑PGL‑1 IgM antibody every During the 5 years observation period only 98 (49%) seroposi‑ year for 5 years with prophylactic treatment. These results tive children had complete data; Nguling 41.8% (33/79) and contradict the findings of Moet et al (18) who showed that Raas 53.7% (65/121). In addition, certain children were lost chemoprophylactic treatment involving SDR had a protec‑ after >1 follow‑up visit and could not be contacted, or their home tive effect only for the initial 2 years. In their initial 2‑year was in a difficult to reach location. Other children moved with follow‑up, rifampicin prevented leprosy, particularly in indi‑ their parents to another city. Overall, the prophylactic drugs viduals aged 10‑14 and 20‑29 years old. The difference in were well tolerated, and few side effects associated with the results may be related to the addition of clarithromycin. The drugs were reported. Notably, after the 5‑years of observation, present study used children who were not contacts of patients there were no children with subclinical leprosy that exhibited with leprosy, and the age range of the recruited cohort was progression to clinical leprosy. The average of anti‑PGL‑1 IgM 7‑14 years old, all from an endemic area of leprosy. However, antibody levels decreased significantly between 2011 and 2015 a previous study by Ghidar et al (31) showed that the addi‑ in Nguling and Raas (from 1,066.72 to 137.44 U/ml and from tion of clarithromycin to a single‑lesion therapy regimen 773.12 to 563.40 U/ml, respectively). Using the antibody titres did not increase cure rates (31). Nevertheless, at the 2‑year classification, the results showed that Nguling was seroposi‑ follow‑up, relapse rates were lower in the patients who tive +2 and Raas had seropositive +1 overall in 2011. received clarithromycin compared with those who did not; The proportion of patients with decreased levels of however, the difference was not significant. M. leprae is an anti‑PGL‑1 IgM antibody in Nguling and Raas was consis‑ obligate intracellular pathogen, which attacks macrophages tently higher than patients with increased levels of anti‑PGL‑1 for replication. A previous study on M. leprae‑infected IgM antibody in all periods, except during 2013‑2014. macrophages showed that invasion interferes with the activa‑ tion of the cell‑mediated immune responses that are required Discussion to attack M. leprae (32). Thus, the bactericidal activity of clarithromycin in macrophages can help improve the host's Elimination of viable M. leprae in a population is a crucial immune response, to reduce the amount of viable M. leprae step to reducing transmission. To be successfully eliminated, and possibility of clinical leprosy. it is important to consider that viable M. leprae are present Previous studies have shown that prophylactic treatment is not only in individuals with clinical leprosy, but also in those very effective when given to the entire population, rather than who have subclinical leprosy. Unfortunately, its presence in only close contacts of patients with leprosy (19,20). This may individuals with subclinical leprosy is often undetected, as be related to the presence of viable M. leprae in undetected the current treatment approaches tend to focus on individuals individuals or in those from untreated areas (20). However, with clinical leprosy. This phenomenon may explain why it has favourable results were obtained in the present study, even proven difficult to completely irradicate leprosy (16). though prophylactic treatment was only administered in a The present study evaluated the efficacy of a chemopro‑ population of elementary school children. phylactic treatment regimen consisting of rifampicin and A previous study in families of patients with leprosy clarithromycin for 5 years. Early detection of subclinical treated with a multidrug treatment (MDT) regimen showed leprosy can be performed using capillary blood and ELISA that several parents were not sufficiently educated. The drugs to determine the titre of anti‑PGL‑1 IgM antibody (21). The in the MDT regimen are usually taken by children, and it has procedure was relatively easy, applicable and convenient for been reported certain children are administered the incorrect 4 ASTARI et al: EVALUATION OF A CHEMOPROPHYLACTIC REGIMEN IN CHILDREN WITH SUBCLINICAL LEPROSY drugs. Therefore, education of chemoprophylactic regimens Competing interests for parents and teachers should be integrated into treatment programmes to promote a positive attitude and supportive The authors declare that they have no competing interests. environment for children (33). The present study has some limitations. Specific doses References of rifampicin and clarithromycin according to age and body weight were not used. The dosage used was based on the 1. WHO: Weekly Epidemiological Report. WHO 91: 405‑420, 2016. age range, as this method is easy but still valid when used in 2. Butlin CR and Saunderson P: Children with leprosy. Lepr Rev 85: 69‑73, 2014. home visits. Treatment with specific doses may help optimize 3. Barreto JG, Guimarães Lde S, Frade MA, Rosa PS and therapy and minimize side effects (28,29). Salgado CG: High rates of undiagnosed leprosy and subclinical In conclusion, the prophylactic treatment of children with infection amongst school children in the Amazon region. Mem Inst Oswaldo Cruz 107 (Suppl 1): S60‑S67, 2012. subclinical leprosy showed good results over the 5 years 4. Oliveira MBB and Diniz LM: Leprosy among children under evaluation period, and the prophylactic regimen used was 15 years of age: Literature review. An Bras Dermato 91: 196‑203, well tolerated. After the 5‑years of observation, there were no 2016. 5. Lourenco DSD, Campelo TA, Cruz GA, De Almeida PC, children with subclinical leprosy that exhibited progression Sa Goncalves HD, Pontes MADA, Lima LNGC and Frota CC: to clinical leprosy, thus this regimen should be considered Detection of subclinical Mycobacterium leprae infection in as a future prophylactic treatment for subclinical leprosy, children, contacts of leprosy cases, Fortaleza‑Ceara', Brazil. Lepr Rev 88: 184‑196, 2017. particularly in children. 6. Adriaty D, Sp CR, Iswahyudi, Wahyuni R, Agusni I and Izumi S: Leprosy transmission in endemic and non‑endemic areas based Acknowledgements on the profile of antibody response of PGL‑1 and PCR detection of Mycobacterium leprae DNA from nasal swab among healthy children of East Java, Indonesia. Infec Dis Rep 12 (Suppl 1): We would like to thank to Mr Sumarsono (Bachelor of Public S8748, 2020. Health) for assisting with the co‑ordination of the present study. 7. Fine PE: Leprosy: The epidemiology of a slow bacterium. Epidemiol Rev 4: 161‑188, 1982. 8. Van Beers SM, de Wit MY and Klatser PR: The epidemiology Funding of Mycobacterium leprae: Recent insight. FEMS Microbiol Lett 136: 221‑230, 1996. 9. Hooij AV and Geluk A: Immunodiagnostic for leprosy. The present study was funded by the East Java Provincial In: International Textbook of Leprosy. Scollard D and Government (grant no. 1.02‑0106‑22‑005‑5‑2). Gillis T (eds). American Leprosy Missions, pp. 2-3, Leiden, The Netherlands, 2016. 10. Moura RS, Calado KL, Oliveira ML and Bührer‑Sékula S: Availability of data and materials Leprosy serology using PGL‑1: A systematic review. Rev Soc Bras Med Trop 41 (Suppl 2): S11‑S18, 2008. The datasets used and/or analyzed during the present study are 11. Araújo S, Lobato J, Reis Éde M, Souza DO, Gonçalves MA, Costa AV, Goulart LR and Goulart IM: Unveiling healthy carriers available from the corresponding author on reasonable request. and subclinical infections among household contacts of leprosy patients who play potential roles in the disease chain of transmis‑ Authors' contributions sion. Mem Inst Oswaldo Cruz 107 (Suppl 1): S55‑S59, 2012. 12. Penna ML, Penna GO, Iglesias PC, Natal S and Rodrigues LC: Anti‑PGL‑1 positivity as a risk marker for the development of LA performed the analysis, and wrote and revised the manu‑ leprosy among contacts of leprosy cases: Systematic review and script based on suggestions from the other authors. NIPS meta‑analysis. PLoS Negl Trop Dis 10: e0004703, 2016. 13. Araujo S, Rezende MM, Sousa DC, Rosa MR, Santos DC, contributed to study design, proofreading and manuscript Goulart LR and Goulart IMB: Risk‑benefit assessment of Bacillus revision. KN, EC, NH, MYL, DA, II, RW, MDA, BHK and Calmette‑Guérin vaccination, anti‑phenolic glycolipid I serology, RIA collected the samples and performed the measurements. and Mitsuda test response: 10‑year follow‑up of household contacts of leprosy patients. Rev Soc Bras Med Trop 48: 739‑745, 2015. IA, SI and CRSP contributed to study design, manuscript 14. Araújo S, Freitas LO, Goulart LR and Goulart IMB: Molecular drafting and revision for intellectual content. All authors have evidence for the aerial route of infection of Mycobacterium leprae read and approved the final manuscript. All authors confirm and the role of asymptomatic carriers in the persistence of leprosy. Clin Infect Dis 63: 1412‑1420, 2016. the authenticity of all the raw data. 15. Goulart IM, Araújo S, Filho AB, de Paiva PH and Goulart LR: Asymptomatic leprosy infection among blood donors may Ethics approval and consent to participate predict disease development and suggests a potential mode of transmission. J Clin Microbiol 53: 3345‑3348, 2015. 16. Reveiz L, Buendía JA and Téllez D: Chemoprophylaxis in contacts All students obtained consent from their parents to participate of patients with leprosy: Systematic review and meta‑analysis. Rev Panam Salud Publica 26: 341‑349, 2009. in the present study, and signed an informed consent form that 17. Barth‑Jaeggi T, Steinmann P, Mieras L, van Brakel W, clearly stated the purpose of the research, the method of sample Richardus JH, Tiwari A, Bratschi M, Cavaliero A, collection, and the lack of risk from medical procedures. Vander Plaetse B, Mirza F, et al: Leprosy Post‑Exposure Prophylaxis (LPEP) programme: Study protocol for evaluating They also received an agreement form and resignation form. the feasibility and impact on case detection rates of contact The present study was approved by the Ethical Committee tracing and single dose rifampicin. BMJ Open 6: e013633, 2016. in Health Research Unit of Dr. Soetomo General Hospital 18. Moet FJ, Pahan D, Oskam L and Richardus JH; COLEP Study Group: Effectiveness of single dose rifampicin in preventing Surabaya, Indonesia (approval no. 30‑511/H3.13/PPd/2012). leprosy in close contacts of patients with newly diagnosed leprosy: Cluster randomised controlled trial. BMJ 336: 761‑764, 2008. Patient consent for publication 19. Bakker MI, Hatta M, Kwenang A, Van Benthem BH, Van Beers SM, Klatser PR and Oskam L: Prevention of leprosy using rifampicin as chemoprophylaxis. Am J Trop Med Hyg 72: Not applicable. 443‑448, 2005. BIOMEDICAL REPORTS 15: 88, 2021 5

20. Smith WC and Aerts A: Role of contact tracing and preven‑ 27. Mieras LF, Taal AT, van Brakel WH, Cambau E, Saunderson PR, tion strategies in the interruption of leprosy transmission. Lepr Smith WCS, Prakoeswa CRS, Astari L, Scollard DM, Rev 85: 2‑17, 2014. do Nascimento DC, et al: An enhanced regimen as post‑exposure 21. Feenstra SG, Pahan D, Moet FJ, Oskam L and Richardus JH: chemoprophylaxis for leprosy: PEP+. BMC Infect Dis 18: 506, 2018. Patient‑related factors predicting the effectiveness of rifampicin 28. MIMS: Drug information. Accessed: April 18, 2021. https://www. chemoprophylaxis in contacts: 6 year follow up of the COLEP mims.com/indonesia. cohort in Bangladesh. Lepr Rev 83: 292‑304, 2012. 29. Grüner N, Stambouli O and Ross RS: Dried blood spots‑preparing 22. Ji B, Jamet P, Perani EG, Bobin P and Grosset JH: Powerful and processing for use in immunoassays and in molecular tech‑ bactericidal activities of clarithromycin and minocycline against niques. J Vis Exp 97: 52619, 2015. Mycobacterium leprae in lepromatous leprosy. J Infect Dis 168: 30. Richardus RA, van der Zwet K, van Hooij A, Wilson L, Oskam L, 188‑190, 1993. Faber R, van den Eeden SJF, Pahan D, Alam K, Richardus JH 23. Walker LL, Van Landingham RM and Shinnick TM: Clarithromycin and Geluk A: Longitudinal assessment of anti‑PGL‑1 serology is bactericidal against strains of Mycobacterium leprae resis‑ in contacts of leprosy patients in Bangladesh. PLoS Negl Trop tant and susceptible to dapsone and rifampin. Int J Lepr Other Dis 11: e0006083, 2017. Mycobact Dis 61: 59‑65, 1993. 31. Girdhar A, Kumar A and Girdhar BK: A randomised controlled 24. Lazo‑Porras M, Prutsky GJ, Barrionuevo P, Tapia JC, trial assessing the effect of adding clarithromycin to rifampicin, Ugarte‑Gil C, Ponce OJ, Acuña‑Villaorduña A, Domecq JP, ofloxacin and minocycline in the treatment of single lesion pauci‑ De la Cruz‑Luque C, Prokop LJ and Málaga G: World health bacillary leprosy in Agra District, India. Lepr Rev 82: 46‑54, 2011. organization (WHO) antibiotic regimen against other regi‑ 32. Yang D, Shui T, Miranda JW, Gilson DJ, Song Z, Chen J, Shi C, mens for the treatment of leprosy: A systematic review and Zhu J, Yang J and Jing Z: Mycobacterium leprae‑infected meta‑analysis. BMC Infect Dis 20: 62, 2020. macrophages preferentially primed regulatory T cell responses 25. Lebel M: Pharmacokinetic properties of clarithromycin: A and was associated with lepromatous leprosy. PLoS Negl Trop comparison with erythromycin and azithromycin. Can J Infect Dis 10: e0004335, 2016. Dis 4: 148‑152, 1993. 33. Lal V, Pal S, Haldar NK, Mandal PK and Srinivas G: What 26. Crowther JR: The ELISA guidebook. Methods Mol Biol 149: parents should know while their child is on MDT: Insights from III‑IV, 1‑413, 2000. a qualitative study in Eastern India. Lepr Rev 85: 81‑84, 2014.