Jpn. J. Infect. Dis., 63, 1-7, 2010 Invited Review Drug Resistance in Leprosy Masanori Matsuoka* Leprosy Research Center, National Institute of Infectious Diseases, Tokyo 189-0002, Japan (Received October 7, 2009) CONTENTS 1. Introduction 2-5. Minocycline 2. Chemotherapy of leprosy 3. Drug susceptibility testing 2-1. Dapsone 3-1. Mouse footpad method 2-2. Rifampicin 3-2. Mutation detection by sequencing 2-3. Ofloxacin 3-3. Mutation detection by DNA microarray 2-4. Clofazimine 4. Perspectives SUMMARY: Leprosy is caused by Mycobacterium leprae. Currently, leprosy control is mainly based on WHO- recommended multi-drug treatment; thus, emergence of drug resistance is a major concern. M. leprae isolates resistant to single and multiple drugs have been encountered. In this review, the history of chemotherapy and drug resistance in leprosy and molecular biological insights for drug resistance are described. New methodolo- gies to test susceptibility to anti-leprosy drugs instead of the traditional mouse footpad method are introduced. Awareness of the need to monitor drug resistance to prevent the spread of resistant cases is emphasized. resistance increased (7) and multi-drug resistant cases 1. Introduction emerged (8–10). The purpose of this review is to describe the Leprosy is a chronic infectious disease caused by an obli- chemotherapy of leprosy, drug resistance to current treat- gate intracellular pathogen Mycobacterium leprae. Newly ments, molecular biological methods to detect drug-resistant detected cases in Japan have markedly decreased during the mutants, and a global strategy to combat the emergence of last two decades. Recently, there have been fewer than 10 drug resistance. cases reported. Most cases are foreigners living in Japan, with about half being Japanese Brazilians (1). In 2008, there were 2. Chemotherapy of leprosy 250,000 new cases globally (2). Although the number has declined rapidly from 2001 to 2005, this reduction is mainly Current treatment of leprosy based on MDT consists of due to a decrease in new cases from India. No change in the dapsone, rifampicin, clofazimine, ofloxacin, and minocycline number of annual new cases outside of India is one enigma as shown in Table 1; however, other antibiotics such as of global leprosy control. streptomycin (11), clarithromycin (12), fluoroquinolones (13– M. leprae possesses neurotropism and causes neuropathy, 15), and ethionamide (16) are also effective in treating lep- which leads to depression of sensation, functional motor rosy. No effective drug was available for leprosy until promin disorder, and deformities. Leprosy shows a wide spectrum of was introduced in the early 1940s. Chaulmoogra oil had been disease types according to the host immune response, from used for leprosy treatment since ancient times, but its efficacy lepromatous type leprosy (LL) to tuberculoid type (TT) lep- was partial and relapse was common (17). For the remainder rosy (3). LL cases develop a Th2 cytokine profile and have a of this section, anti-leprosy drugs are introduced and resistance high bacillary load. In contrast, TT cases develop a Th1 to drugs currently used in MDT is described. cytokine profile and few bacilli are observed. Since no vaccine 2-1. Dapsone is available, early diagnosis and treatment is the basic strat- The first successful chemotherapy using promin for lep- egy for leprosy control. Treatment is based on multi-drug rosy was reported in 1943 (18) at a leprosarium in Carville, therapy (MDT), recommended by WHO (4,5). In this regi- Louisiana, USA. Promin (diamiono-azobenzene 4´-sulfona- men, leprosy is classified into one of three types, including mide) was introduced in Japan in 1947. Patients were intra- multi bacillary (MB), paucibacillary (PB), and single-lesion venously administrated 5 ml of 30% promin solution. A more PB (SLPB) according to the number of skin lesions (Table effective sulfone, dapsone (4,4´-diaminodiphenyl sulphone: 1). MB and PB patients are treated for 1 year and 6 months, DDS), replaced promin 6 years later. Dapsone is still a funda- respectively. Although MDT is an effective leprosy treatment mental anti-leprosy compound even in the MDT era. Dapsone and has reduced prevalence in many areas, there is drug inhibits folic acid synthesis by competitive inhibition and is resistance to the individual MDT components. The first clini- bacteriostatic. cally suspected drug-resistant case to dapsone was reported Dapsone, an analogue of p-aminobenzoic acid (PABA), in 1953 (6). Soon after introducing the drug, the incidence of targets dihydropteroate synthase (DHPS), which is encoded by folP1 and is involved in folic acid synthesis. A relation- *Corresponding author: Mailing address: Leprosy Research Cen- ship between DHPS mutations and dapsone resistance has ter, National Institute of Infectious Diseases, 4-2-1 Aobacho, been demonstrated. Missense mutations at codon 53 (ACC) Higashimurayama, Tokyo 189-0002, Japan. Tel: +81-423-91- or 55 (CCC) coding threonine or proline in folP1 confer dap- 8211, Fax: +81-423-94-9092, E-mail: [email protected] sone resistance (19,20). Mutations detected in folP1, of which 1 resistance to dapsone was confirmed by the mouse footpad should be reevaluated by the mouse footpad test. assay, include codon 53 ACC (Thr) to ATC (Ile), GCC (Ala), Although the first dapsone-resistant cases were reported GTC (Val), AGA (Ala) or AGG (Arg) (9,20–24), and codon in 1953, no methodology to confirm resistance was available 55 CCC (Pro) to CTC (Leu), CGC (Arg), or TCC (Ser) until the mouse footpad assay was developed (26). Using this (10,19,21–25) (Fig. 1). Drug susceptibility in the mouse test, three suspected clinical isolates of dapsone resistance footpad assay and the sequence of isolates examined in our were shown to be dapsone-resistant by growth in the footpad laboratory are shown in Table 2. No mutation was detected of mice given a diet containing 0.1% dapsone (27). The in codon 53 or 55 in folP1 in 88 isolates and 8 isolates of number of dapsone-resistant cases increased after dapsone low-degree dapsone resistance (19,22–24), which is defined monotherapy during the 1960s and 1970s (7,28). Primary by the growth in the mouse footpad fed diets with different dapsone resistance, i.e., resistance in a patient without previ- concentrations of dapsone. Two exceptional cases were found: ous administration of anti-leprosy drugs, and secondary drug one dapsone-resistant isolate with an intermediate degree resistance have been detected in many areas (23,24). harbored no mutation and one isolate with low-degree resist- 2-2. Rifampicin ance harbored the mutation GCC at codon 53. Other isolates Rifampicin 3-[[(4-methyl-1-piperazinyl)-imino]-methyl] is with GCC at codon 53 were dapsone-resistant to a high de- currently a key bactericidal antibiotic for leprosy treatment. gree (23), and all other resistant isolates with an intermediate A high bactericidal effect was shown experimentally and clini- degree of resistance harbored mutations at codon 53 or 55 cally in the 1970s (29–32) leading to the introduction of (10,23); therefore, the susceptibility of these two isolates rifampicin for leprosy treatment. A single dose of 1,200 mg Table 1. WHO/MDT regimen for adults Criteria Dose Duration MB >6 skin lesions Rifampicin: 600 mg once a month 12 months Positive bacterial index Dapsone: 100 mg daily Clofazimine: 300 mg once a month and 50 mg daily PB 2–5 skin lesions Rifampicin: 600 mg once a month 6 months Negative bacterial index Dapsone: 100 mg daily SLPB Single skin lesion Rifampicin: 600 mg Single dose Negative bacterial index Ofloxacin: 400 mg Minocycline: 100 mg MDT, multi-drug therapy; MB, multi bacillary; PB, paucibacillary; SLPB, single-lesion PB. Table 2. Drug susceptibility and sequence of isolates examined by the mouse footpad method Dapsone Rifampicin Quinolone Isolate folP1 MFP* rpoB MFP gyrA MFP Hoshizuka-4 55:Pro(CCC) Resistant 425:Ser(TCG) Resistant 91:Ala(GCA) Resistant → Ser(TCC) Intermediate → Leu(TTG) → Val(GTA) Zensho-4 53:Thr(ACC) Resistant 425:Ser(TCG) Resistant 91:Ala(GCA) Resistant → Ile(ATC) High → Leu(TTG) → Val(GTA) Airaku-2 55:Pro(CCC) Resistant 425:Ser(TCG) Resistant No mutation Susceptible → Leu(CTC) High → Leu(TTG) Zensho-5 55:Pro(CCC) Resistant 425:Ser(TCG) Resistant No mutation Susceptible → Leu(CTC) High → Leu(TTG) Kusatsu-6 55:Pro(CCC) Resistant 410:Asp(GAT) Resistant No mutation Susceptible → Leu(CTC) High → Tyr(TAT) Kusatsu-3 53:Thr(ACC) Resistant No mutation Susceptible No mutation Susceptible → Ile(ATC) High Zensho-2 55:Pro(CCC) Resistant No mutation Susceptible No mutation Susceptible → Leu(CTC) High Amami-1 55:Pro(CCC) Resistant No mutation Susceptible No mutation Susceptible → Leu(CTC) High Zensho-9 No mutation Susceptible 420:His(CAC) Resistant No mutation Susceptible → Try(TAC) Kanazawa No mutation Susceptible No mutation Susceptible No mutation Susceptible Izumi No mutation Susceptible No mutation Susceptible No mutation Susceptible Keifu-4 No mutation Susceptible No mutation Susceptible No mutation Susceptible Ryukyu-2 No mutation Susceptible No mutation Susceptible No mutation Susceptible Kyoto-2 No mutation Susceptible No mutation Susceptible No mutation Susceptible Kitazato No mutation Susceptible No mutation Susceptible No mutation Susceptible Thai-53 No mutation Susceptible No mutation Susceptible No mutation Susceptible Korea 3-2 No mutation Susceptible No mutation Susceptible No mutation Susceptible Indonesia-1 No mutation Susceptible No mutation Susceptible No mutation Susceptible *MFP, mouse footpad testing. 2 to rifampicin (40). Resistance to backbone antibiotics for lep- rosy treatment threatens leprosy control, and this concern led to the introduction of MDT in the 1980s. 2-3. Ofloxacin Ofloxacin (4-fluoroquinolone) is a moderate bactericidal antibiotic for M. leprae. Its bactericidal activity for M. leprae was first demonstrated in 1986 by the mouse footpad method and subsequently by a clinical trial (13,41,42). Ofloxacin binds to the A subunit of DNA gyrase (gyrA) and inhibits DNA replication.