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Drug Resistance in Leprosy

Drug Resistance in Leprosy

Jpn. J. Infect. Dis., 63, 1-7, 2010

Invited Review in Masanori Matsuoka* Leprosy Research Center, National Institute of Infectious Diseases, Tokyo 189-0002, Japan (Received October 7, 2009) CONTENTS 1. Introduction 2-5. 2. Chemotherapy of leprosy 3. Drug susceptibility testing 2-1. 3-1. Mouse footpad method 2-2. 3-2. Mutation detection by sequencing 2-3. 3-3. Mutation detection by DNA microarray 2-4. 4. Perspectives

SUMMARY: Leprosy is caused by 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 . 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 such as of global leprosy control. (11), (12), fluoroquinolones (13– M. leprae possesses neurotropism and causes neuropathy, 15), and (16) are also effective in treating lep- which leads to depression of sensation, functional motor rosy. No effective drug was available for leprosy until 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 (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 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 (13,41,42). Ofloxacin binds to the A subunit of DNA gyrase (gyrA) and inhibits DNA replication. Association between mutations within the highly conserved region of gyrA, coded by the gyrA gene, and quinolone resistance was revealed in most resistant strains of mycobacterium (43,44). The first ofloxacin-resistant M. leprae was found in 1994 (8). Two other cases of ofloxacin resistant M. leprae from Japanese relapsed cases were re- ported in 2000 and 2003 (9,10). Four other possible ofloxacin- Fig. 1. Sequence detected in wild type and drug-resistant mutants. Muta- resistant cases have been found (21). Three isolates confirmed tions in folP1, rpoB, and gyrA of M. leprae confer resistance to dap- to be ofloxacin resistant harbored the mutation Ala-Val (GCA- sone, rifampicin, and quinolone, respectively. (A) folP1: codon 55 GTA) at codon 91 (8–10) in the gyrA gene (Fig. 1). One iso- (CCC: Pro → CTC: Leu); (B) rpoB: codon 425 (TCG: Ser → TTG: late in which the susceptibility could not be examined by the Leu); (C) gyrA: codon 91 (GCA: Ala → GTA: Val). mouse footpad method had the mutation Gly-Cys (GGA- TGC) at codon 89 (21). Two other amino acid changes, Ser at or a daily dose of 600 mg for 3 days kills bacilli in patients, 91, and Asp at 94 (same codon numbers as M. ), and no bacillary growth is shown in mice inoculated with in gyrA of M. tuberculosis have been associated with patient bacilli (32). quinolone resistance (45). It seems that mutations at codons Rifampicin targets the beta (β) subunit of RNA polymerase, 89, 92, and 95 in gyrA of M. leprae also confer quinolone which is encoded by rpoB. Rifampicin binding to the β subunit resistance. No quinolone-resistant isolate was detected from inhibits DNA-dependent mRNA . A correlation 78 isolates in Myanmar and 96 isolates in Cebu, Philippines between rifampicin resistance and mutations at highly con- (24). served regions in the rpoB gene has been shown. Isolates 2-4. Clofazimine confirmed to be rifampicin-resistant harbor missense muta- Clofazimoine is [(3-p-chloroanilino)-10-(p-chlorophenyl)- tions at codon 407, 410, 420, 425, and 427 (8–10,25,33–36). 2,10-dihydro-2-(isopropylimino) ], and is bacteri- In addition, one isolate had a 6-bp insertion in codon 409 cidal for M. leprae. The mechanism of action is not fully (34). Various mutations detected from isolates confirmed to elucidated; however, a possible bactericidal mechanism be rifampicin resistant include codon 407 CAG (Gln) to GTG through the binding of GC-rich domains is suggested (46). (Val) (33), codon 410 GAT (Asp) to TAT (Tyr) (25), codon No molecular background for drug resistance to clofazimine 420 CAC (His) to GAC (Asp) (33,34), codon 425 TCG (Ser) is known. Clofazimine was first used for leprosy treatment in to TTG (Leu) (8–10,21,33,35,36), ATG (Phe) (35), TTC (Met) 1962 (47). It is anti-inflammatory and is also used to control (35), and codon 427 CTG (Leu) to GAG (Val) (33) (Fig. 1). the type 2 reaction ( leprosum: ENL) in Of 29 mutants, 22 isolates had the mutation TTG at codon MDT. Although clofazimine has been used for leprosy treat- 425. The high frequency of this mutation is similar to the ment for over four decades, reported drug resistance is rare frequency of this mutation in Mycobacterium tuberculosis (48–51). (35,36). In addition, a codon 416 amino acid substitution of 2-5. Minocycline Ser to Cys (TCG-TGT) was found, but results of the mouse Minocycline (7-dimethylamino-6-demethyl-6-deoxy- footpad susceptibility test were unavailable for this isolate. ) is the only tetracycline group active against M. tuberculosis with this mutation is resistant to rifampicin M. leprae. Efficacy of minocycline against M. leprae was (36,37). Therefore, it could be concluded that this mutation confirmed in 1987 (52). It is bactericidal and its activity is confers rifampicin resistance to M. leprae. Although the additive when combined with other anti-leprosy drugs (53,54). mutation Gly408Asp was detected, this mutation was detected Minocycline is used with rifampicin and ofloxacin in MDT together with a mutation at codon 420 that is known to con- only for the SLPB cases. inhibit protein synthe- fer rifampicin resistance. Thus, it was unclear whether the sis by binding to the 30S ribosomal subunit, blocking the Gly408Asp mutation is related to rifampicin resistance. A binding of aminoacyl transfer RNA to the messenger RNA total of 108 rifampicin-susceptible strains determined by the ribosomal complex (55). To date, no minocycline-resistant mouse footpad test did not contain any mutations between cases are known. codons 407 to 427 (24,33,35,36). Treatment with inappropriate regimens is one main reason Prior to the advent of MDT, rifampicin had been used as drug-resistant strains develop and relapses with a resistant monotherapy or in combination with dapsone. The first strain occur. There were no authorized MDT regimens for rifampicin-resistant case was shown in an isolate from a leprosy treatment in Japan until 1997 (56), and guidelines relapsed patient previously treated by dapsone in 1976 (38). were provided by the Japanese Leprosy Association in 2000 A susceptibility test for 45 relapsed cases treated previously (57). Such inadequate conditions in defining treatment might by rifampicin alone or in combination with dapsone showed be the reason for the high prevalence of drug resistance among that nine cases were rifampicin resistant (39). Another study relapsed cases in Japan (21). revealed that 19 out of 35 relapsed cases were resistant to both dapsone and rifampicin and three isolates were resistant

3 Over the past two decades, mutations leading to drug resist- 3. Drug susceptibility testing ance to dapsone, rifampicin, and quinolones have been re- 3-1. Mouse footpad method vealed. Based on this knowledge, DNA-based assays for M. leprae has still not been cultivated in vitro, and sus- detecting drug resistant M. leprae have been developed. ceptibility testing was hampered until the development of the As mentioned above, missense mutations within a limited mouse footpad assay (26). The first dapsone-resistant case region in folP1, rpoB, and gyrA are in concordance with was confirmed with this method in 1964 (27). Mouse foot- resistance to dapsone, rifampicin, and quinolones confirmed pad testing is still the gold standard for drug susceptibility by mouse footpad testing. These regions are drug resistance- in leprosy. A bacillary suspension is prepared from a lesion determining regions (DRDR). Results of mouse footpad test- biopsy sample containing bacilli. Mice are inoculated with ing and sequence analysis are shown in Table 2. Resistance 5,000–10,000 bacilli into the hind footpad and the bacillary to dapsone is classified into three grades according to bacil- number reaches 105 to 106 approximately 25 to 30 weeks lary growth in the mouse footpad assay with different con- after inoculation. Mice inoculated with M. leprae are divided centrations of dapsone (Table 3). No mutations are detected into groups of 10 to 20 mice. One group is a control, and the in folP1 from 10 isolates with a low degree of dapsone resist- remaining groups are administered drugs. Drugs are admin- ance (23,24). Though Shepard et al. (59) found that M. leprae istered per os, either incorporated into the mouse diet (Table obtained from untreated leprosy patients in an earlier era were 3) or by gavage (8–10,58). When bacillary growth is recog- consistently inhibited by 0.0001% dapsone in the diet, Rees nized in control mice at about 25–30 weeks, mice are sacri- (60) found some isolates that were not inhibited at this ficed and bacillary growth is compared between the control dapsone concentration. Dapsone resistance in the mouse at a group and each group of treated mice. An isolate is regarded concentration of 0.0001% is not associated with folP1 muta- as drug resistant when bacillary growth is present in the mouse tions. Such cases have no clinical significance, since admin- footpad. Results of bacillary growth for isolates resistant to istration of 0.0001 g dapsone per 100 g mouse diet would be dapsone, rifampicin, and quinolones are shown in Fig. 2. similar to humans receiving 1 mg dapsone daily (61). The 3-2. Mutation detection by sequencing usual dosage of dapsone in MDT is 100 mg daily. The muta- Susceptibility testing by the mouse footpad method is cum- tion detection in DRDR indicated above predicts drug resist- bersome and time-consuming, is not applicable to many ance to these three drugs. strains, and does not meet the needs of clinicians. The method Since some isolates do not multiply in control mouse foot- requires viable bacilli and chilled samples to be sent to the pads, their drug susceptibility cannot be determined by this laboratory, which is usually far from the field, within a week. assay. By contrast, DNA sequencing of PCR products is in- dependent of the viability of M. leprae. Among 83 isolates, Table 3. Criteria for diagnosing drug resistance 46 isolates could be tested for drug susceptibility by the mouse in the mouse footpad method while 79 isolates could be sequenced and Drug Concentration in the diet (%) their susceptibility determined (33). Results reveal the use- fulness of molecular biological methods for susceptibility Dapsone 0.0001; 0.001; 0.01* testing. To prevent the spread of drug-resistant isolates, com- Clofazimine 0.0001–0.001 prehensive data on the magnitude of resistance is essential. Rifampicin 0.003–0.01 Sequencing PCR products could be applied to a large number Ofloxacin 0.15 of samples. The level of drug resistance in three South Asia 0.02 countries was analyzed using this method; that study was the Clarithromycin 0.03 first in which sequencing analysis was applied to a large Minocycline 0.08 number of samples (24). Isolates with primary or secondary *Low, intermediate or high level of resistance, resistance to dapsone and rifampicin were detected in Indone- respectively. sia and Myanmar. Primary and secondary daspone resistance

Table 4. Prevalence of drug resistance in M. leprae isolates from Asian countries Indonesia (North Maluku and North Sulawesi) New or recent case Relapse case Dapsone Rifampicin Ofloxacin Dapsone Rifampicin Ofloxacin 1/121 4/121 N.D 1/10 2/10 N.D (0.8%) (3.3%) (10%) (20%) Myanmar (Yangon) New or recent case Relapse case Dapsone Rifampicin Ofloxacin Dapsone Rifampicin Ofloxacin 4/54 1/54 0/54 2/24 2/24 0/24 (7.2%) (1.8%) (8.3%) (8.3%) Philippines (Cebu) New or recent case Relapse case Dapsone Rifampicin Ofloxacin Dapsone Rifampicin Ofloxacin Fig. 2. Bacillary growth after 30 weeks in the footpad of mice admin- 2/77 0/77 0/77 5/19 0/19 0/19 istered anti-leprosy drugs. Circles indicate bacillary number in the (2.6%) (26%) footpad of each mouse. DDS, dapsone. Reprinted with permission from (10). Modified with permission from (24).

4 A Drug Gene Codon no. Capture probes for wild type Capture probes for mutants

Dapsone folP1 53 FW1:GTGGCGAATCGACCCGG FM1:TGGCGAATCGGCCCGG FM2:TGGCGAATCGGTCCGG FM3:TGGCGAATCGATCCGG FM4:GGCGAATCGAGGCGG FM5:TGGCGAATCGAGACGG 55 FW2:CGGCCCGGTGCCATTA FM6:GACCGGTCCGGTGCC FM7:GACCGGCGCGGTGCC FM8:GACCGGCTCGGTGCC Rifampicin rpoB 407 RW1:AGCTGTCGCAGTTCATG RM1:AGCTGTCGGTGTTCAT 410 RW2:TTCATGGATCAGAA RM2:TTCATGAATCAGAACAA RM3:TTCATGTATCAGAACAA 420 RW3:CCTGACCCACAAGCGC RM4:GCCTGACCTACAAGCGC RM5:GCCTGACCGACAAGCGC 425 RW4:CGCCGACTGTCGGCGCTG RM6:CCGACTGATGGCGC RM7:GCCGACTGTTGGCG RM8:GCCGACTGTTCGCG 427 RW5:GCGCTGGGGCCCGGGTG RM9:GGCGCCGGGGCCCGGGTG Ofloxacin gyrA 89 GW1:ATCCGCACGGCGACGCA GM1:ATCCGCACTGCGACGCA 91 GW2: CGGCGACGCATCGATTT GM2: CGGCGACGTATCGATTT Positive hybridization control in gyrA GP:GGACCGTAGCCACGCTAA Negative hybridization control in gyrA GN:GGACCGTCATCACGCTAA

B ddapsone fildfield rifampicin if ii fildfield

bion FW1 FM1 FM2 FM3 FM4 FM5bion RW1 RM1 bion

FW2 FM6 FM7 FM8 RW2RM2 RM3

GW1 GM1 RW3 RM4 RM5 ofloxacin field GW2 GM2 RW4RM6 RM7 RM8

bionGP GN control field bionRW5 RM9 bion

Fig. 3. Development of a drug susceptibility screen for leprosy. (A) Oligonucleotide sequences used in the screen are shown. Codons related to drug resistance are underlined. Oligonucleotide GP, positive control for PCR amplification and hybridization. GN, negative control for hybridization. (B) Schematic representation of oligonucleotide array on the LDS-DA. Black circles represent spots with biotin that are landmarks for conjugate reaction controls. Gray circles are wild-type spots. White circles are mutant spots. The region with oligonucleotides designated FW- and FM- detects dapsone resistance (the dapsone field); the region designated GW- and GM- is the ofloxacin field; the region designated GP- and GN- is the control field; and the region designated RW- and RM- is the rifampicin field. Reprinted with permission from (62).

robust method to detect drug resistance, but implementation of sequencing is not easy in many developing countries where the prevalence of leprosy is still high. To overcome this dis- advantage, a simple method, leprosy drug susceptibility-DNA microarray (LDS-DA), to detect mutations was exploited (63). A series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB, and gyrA genes for dapsone, rifampicin, and ofloxacin resistance, respectively, are selected and fixed on a glass slide as capture probes (Fig. 3). Biotin- labeled PCR products are simultaneously amplified in a tube and hybridized with a probe corresponding to each mutation (Fig. 4). The usefulness and validity of the methods has been evaluated by applying them in the Philippines and Myanmar. The high concordance of results obtained by this method in the two countries with those of nucleotide sequencing is Fig. 4. Signal obtained with a susceptible strain Thai-53 and Zensho-4 that has the following mutations: ATC (from ACC) in codon 53 in shown. The method is feasible for testing drug susceptibility folP1, TTG (from TCG) in codon 425 in rpoB, and GTA (from GCA) in at a laboratory close to the field. These obtained data might codon 91 in gyrA. PCR products include drug resistance determining be beneficial for both clinicians and patients by avoiding use- regions for three genes were amplified simultaneously in a tube. less drug dosages. was detected in Cebu, Philippines, but no rifampicin-resist- ant cases were found. Fortunately, drug resistance in the sur- 4. Perspectives veyed areas is still rare (Table 4). Current leprosy control solely depends on MDT, which 3-3. Mutation detection by DNA microarray uses five drugs. Resistance to dapsone, rifampicin, and Direct sequencing of PCR products covering DRDR is a ofloxacin can be detected by molecular techniques. Although

5 resistance to clofazimine and minocycline is not detected by R.H. (ed.), Leprosy. Churchill Livingston, Edinburgh. sequence analysis of specific genes, the frequency of drug 18. Faget, G.H., Pogge, R.C., Johansen, F.A., et al. (1943): The promin treatment of leprosy: a progress report. Public Health Rep., 58, 1792– resistance to clofazimine is negligible, and no resistance to 1741. minocycline has been found. Therefore, most MDT drug- 19. Kai, M., Matsuoka, M., Nakata, N., et al. (1999): Diaminodiphenylsulfone resistant cases are detectable by sequencing. Emergence of resistance of Mycobacterium leprae due to mutations in the drug resistance must lead to the implementation of interven- dihydropteroate synthase gene. FEMS Microbiol. Lett., 177, 231–235. 20. Williams, D.L., Spring, L., Harris, E., et al. (2000): Dihydropterote tion programs. synthase of Mycobacterium leprae and dapsone resistance. Antimicrob. 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