jcm accepts, published online ahead of print on 11 april...
TRANSCRIPT
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Running title: leprosy relapse and mutations 1
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DRUG AND MULTIPLE-DRUG RESISTANCE AMONG MYCOBACTERIUM LEPRAE 3
ISOLATES FROM BRAZILIAN RELAPSED LEPROSY PATIENTS 4
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Adalgiza da Silva Rocha1, Maria das Graças Cunha2, Lucia Martins Diniz3, Claudio 6
Salgado4, Maria Araci P. Aires5, José Augusto Nery6 Eugênia Novisck Gallo6, Alice 7
Miranda6, Monica M. F. Magnanini7, Masanori Matsuoka8, Euzenir Nunes Sarno6, Philip 8
Noel Suffys1 and Maria Leide W. de Oliveira7 9 10
1 Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Fiocruz, 11
Rio de Janeiro, RJ, Brazil 12
[email protected]; [email protected]; 13
2 Tropical Dermatology Foundation Alfredo da Matta (FUAM), Manaus, Amazonas, Brazil- 14
3 Dermatology Service, Santa Casa de Misericordia (EMESCAM), Vitória, Espirito Santo, Brazil 16
4 Laboratory of Pathology, Reference Center Marcelo Candia-SES, Federal University of Para 18
(UFPA), Marituba, Para, Brazil 19
5 Reference Center in Sanitary Dermatology D. Lebanon (CDERM), Fortaleza, SES, Ceara, 21
Brazil [email protected] 22
Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.06561-11 JCM Accepts, published online ahead of print on 11 April 2012
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6 Leprosy Laboratory, Oswaldo Cruz Institute, Fiocruz, RJ, Brazil- [email protected]; 23
[email protected]; [email protected]; [email protected] 24
7 Training Center on Dermatology, University Hospital HUCFF and Public Health Institute, 25
Federal University of Rio de Janeiro, RJ, Brazil 26
[email protected]; [email protected]; [email protected] 27
8 Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan 28
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Corresponding author: Maria Leide W. de Oliveira: [email protected]/[email protected] 31
HUCFF/UFRJ- Tel / FAX: 55-21-25622580/25622785 32
Rua Rodolpho P Rocco 255, Ilha do Fundão, 5º andar, Sala B11, Dermatologia 21.941913 33
RJ 34
35
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ABSTRACT 37
Skin biopsy samples from 145 relapse leprosy cases and from five different regions in 38
Brazil, were submitted to sequence analysis of part of the genes associated with Mycobacterium 39
leprae drug resistance. Single nucleotide polymorphisms in these genes were observed in M. 40
leprae from four out of 92 cases with positive amplification (4.3%) and included a case with a 41
mutation in rpoB only, another sample with SNPs in both folP1 and rpoB and two cases showing 42
mutations in folP1, rpoB and gyrA, suggesting the existence of multi-drug resistance (MDR). 43
The nature of the mutations were as reported in earlier studies, being CCC to CGC in codon 55 44
in folP (Pro to Arg), while in the case of rpoB, all mutations occurred at codon 531, two being a 45
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transition of TCG to ATG (Ser to Met), one TCG to TTC (Ser to Phe) and one TCG to TTG (Ser 46
to Leu). The two cases with mutations in gyrA changed from GCA to GTA (Ala to Val) in codon 47
91. The median time from cure to relapse diagnosis was 9.45 years but was significantly shorter 48
in patients with mutations (3.26 yrs; p = 0.0038). Over 70% of the relapses were multibacillary, 49
including three of the mutation carrying cases; one MDR relapse patient was paucibacillary. 50
51
Key words: leprosy, relapse, sequencing, multidrug resistance 52
53
INTRODUCTION 54
There is no doubt about the efficiency of the currently used multi-drug therapy (MDT) 55
scheme for treatment of leprosy, as demonstrated by the strong decrease in disease prevalence 56
since its implementation and the low number of reported relapse cases (18). However, there is a 57
scarcity of indebt studies on relapse occurrences in recent decades (27). As is known, 58
differentiating diagnosis of relapse and reactional states poses some difficulties in the field, being 59
responsible for under- or over-diagnosis of both disease stages. This is important because 60
undiagnosed relapse cases could contribute to continuing disease transmission. In addition, 61
hardly any data exists on the contribution of emergence of drug resistant strains of 62
Mycobacterium leprae to leprosy relapses. 63
Diamino-diphenyl sulfone (DDS), also called dapsone, was the first drug effective 64
against leprosy worldwide and the first cases of resistance to dapsone were detected in 1964 and 65
involved two single nucleotide polymorphisms (SNPs) in the gene folP1, located in codons 53 66
and 55 (9, 10, 14, 29). Rifampicin is the key component of the standard multi-drug regimen used 67
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for treatment of leprosy and it has been shown that PCR-based DNA sequence analysis of the 68
rpoB gene of M. leprae was in full concordance with rifampicin susceptibility testing in the 69
mouse footpad system (17, 30). In addition to dapsone and rifampicin, ofloxacin is also used for 70
leprosy treatment and is a quinolone with action mechanism based on interaction with DNA 71
gyrase (2) and SNPs in gyrA and gyrB confer resistance or hypersensitivity to quinolones (15). 72
Although there is not yet an official definition of what is multi-drug resistance (MDR) in leprosy, 73
in parallel with tuberculosis, we adopt this terminology when we encounter resistance to 74
rifampicin and one more drug of the standard MDT regimen. 75
Emerging drug resistance has been observed among many diseases caused by bacteria, 76
and this could pose a challenge for the treatment of leprosy, a neglected disease with a minimal 77
therapeutic arsenal (22). Brazilian studies show relapse rates below 1% (12, 26) and drug 78
resistance does not seem to be an important problem in the country (11, 21). Nonetheless, a pilot-79
project for optimal detection of relapse and the contribution of drug resistance among leprosy 80
patients of five states in Brazil was started in 2006 (26), in parallel with the initiative of the 81
World Health Organization (WHO) to perform global surveillance of drug resistance in leprosy 82
in 2008 (36). 83
84
METHODS 85
Study design and patients. 86
A prospective study for detection of relapse in leprosy patients was designed for more 87
accurate determination of the frequency of relapse by drug resistance among Brazilian leprosy 88
patients, based on evaluation of DNA sequencing in samples from 145 leprosy patients, collected 89
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during 2006 to 2008, in five highly endemic states for leprosy, including of Rio de Janeiro, 90
Espírito Santo, Amazonas, Pará and Ceará (26). All patients were examined by experienced 91
dermatologists in six state reference units, in order to guarantee the quality and uniformity of 92
these procedures. Leprosy relapse detection was based on standardized and optimized diagnostic 93
procedures and criteria for definition of relapse (4) and with inclusion criteria being suffering 94
from active clinical lesions of leprosy, as confirmed by smears and histopathological exams; 95
being considered cured from the first disease course after having been submitted to the Brazilian 96
Leprosy Program treatment regimens. Regarding the official treatment regimens from the 97
National Leprosy Program, it is necessary to clarify that Brazil, before adopting the WHO MDT 98
treatment schemes in 1986 (24 doses), used a scheme called “DNDS” that consisted of 90 daily 99
doses of 600 mg of rifampicin, followed by daily doses of 100 mg dapsone monotherapy, up to 100
five years and until slit skin smears became AFB negative. For each relapse case, a control case, 101
being a new leprosy case of the same sex, clinical form and municipality of residence and 102
belonging to the same treatment cohort, was selected from the SINAN (National Information 103
System for Notification Diseases) and enrolled for clinical and laboratory examinations 104
This study was approved by the Ethical Committee of Research of the Federal University 105
of Rio de Janeiro (HUCFF/UFRJ, nº 019/06). Written consent was obtained from individual 106
subjects by signing a standard Brazilian form before being admitted in the study. The 107
epidemiologic, clinical and demographic data collected from each participant center were stored 108
and analyzed at the UFRJ, using Strata software/9.0. 109
Slit skin smear and histopathology of the skin biopsies. 110
As part of the diagnostic procedure, slit skin smear samples were collected from four 111
different body sites at the time of diagnosis of disease relapse, and a skin biopsy, according to 112
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standard recommendations (Brasil, 2010). After being cut in half, one part of the skin biopsy was 113
prepared for histopathology exam, and the other half immersed in 70% ethanol for genetic 114
analysis. In order to standardize the histopathology procedure and reporting of results, a 115
consensus meeting was held with the histopathologists from the participating reference centers 116
and a standard protocol was elaborated. 117
Extraction of nucleic acids. 118
For extraction of nucleic acids, the ethanol was removed from the biopsy, and the latter 119
rehydrated, cut into small pieces and submitted to DNA extraction and purification using the 120
Qiagen kit DNeasy Blood & Tissue (Invitrogen do Brasil). In brief, 180 µl of ATL buffer and 20 121
µl of proteinase K from the kit were added to the biopsy, mixed by vortex and after overnight 122
incubation at 56ºC, DNA was purified using spin-column from the kit as described by the 123
manufacturer. 124
Amplification and sequencing analysis of part of rpoB, folP1, gyrB and gyrA. 125
Part of the genes rpoB, folP1 and gyrA were analyzed by direct sequencing of polymerase 126
chain reaction (PCR) products generated using conditions described previously, using 127
amplification primers MrpoBF and MrpoBR (31), folP1F and folP1R (38) and gyrANF and 128
gyrANR (5, 8, 23), and using touch-down amplification conditions described before (8). Each 129
PCR reaction contained at least one negative control and after verification of PCR product 130
quantity and quality on 3% agarose gel, amplicons were purified using the ChargeSwitch PCR 131
Clean-Up Kit (Invitrogen do Brasil) and sequenced using the same primers as those for 132
generating the PCR fragment of each gene, using the ABI Big Dye 3.1 Terminator Ready 133
Reaction Kit (Applied Biosystems do Brasil). For characterization of the gyrA SNP at position 134
297, we followed the approaches described previously (8). Sequences were generated on an ABI 135
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3730 Genetic Analyzer (Applied Biosystems) and compared with the M. leprae sequences 136
NC002677 and z14314 (rpoB), AL023093 (folP1) and NC002677 (gyrA), available at GenBank 137
(http://www.ncbi.nlm.nih.gov/sites/entrez/) and for SNP analysis, sequences were introduced 138
into SeqScape. Control DNAs were purified M. leprae NHDP-63 (kindly donated by Dr Patrick 139
Brennan, Colorado State University, USA) and the plasmids folP101, 102 and 103 (gift from Dr. 140
Dianna Williams, Louisiana State University, USA). Following the recommendations of the 141
“WHO Global Surveillance of Drug Resistance in Leprosy Protocol”, samples with mutations 142
suggestive for drug resistance as determined at FIOCRUZ were send for blind sequence 143
evaluation to Dr. Masanori Matsuoka at the Leprosy Research Center National Institute of 144
Infectious Diseases, Tokyo, Japan. 145
In order to verify the presence of inhibitors in the processed biopsy samples, 23 biopsy 146
samples that gave no PCR product in the gyrA system were submitted to a reconstitution 147
experiment to verify the presence of eventual PCR inhibitors. For this, these samples were 148
submitted to the PCR using the same conditions as described above, except for the addition of 149
1.5 ng of NHDP-63 DNA to each PCR reaction. For evaluation of inhibition, the PCR signal of 150
reactions with biopsy sample was compared to that of reconstituted samples without biopsy 151
sample and two positive controls (without reconstitution) as for the earlier PCR experiments. 152
We used three interpretation criteria, being either (i) similar or (ii) less signal than the control 153
samples or (iii) no amplification at all. 154
155
RESULTS 156
General patient data. 157
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Clinical data confirmed that 145 patients suffered from leprosy relapse and their 158
characteristics are summarized in Table 1. All these patients presented the inclusion criteria, 159
having been considered cured after completing the official treatment regimen (Brazil/DNDS or 160
WHO/MDT), having developed a second course of active leprosy disease, as confirmed by 161
baciloscopic and histopathological examination, allowing also the classification of the clinical 162
form. Most cases (70%) were multibacillary (MB) while the rest were paucibacillary (PB), 163
among the latter, the majority (88%) being Borderline Tuberculoid. The Bacilloscopy Index (BI) 164
of the MB cases ranged between 0.25 and 6.0, with an average of 2.85. The average incubation 165
period from cure to relapse diagnosis was 9.45 years and ranged between 1.5 and 25 years, and 166
was significantly shorter in the four resistant cases (3.26 yrs; p = 0.0038), ranging between one 167
month and 6.6 years. In addition, two of these cases had been submitted to more than one 168
treatment regimen. Gender analysis showed that males (72.4%) were more affected than females 169
(26.4%) and the median age of all cases at time of diagnosis of relapse was 47.5, ranging from 170
13 to 96 years (Table 1). 171
Upon analyzing treatment regimens, we observed that most of the MB first disease cases 172
had been treated with the MDT/WHO scheme, having completed either 24 or 12 doses, as 173
adopted by the National Program; some MB cases however, instead of having received 12 doses, 174
had been submitted to either (i) a number of doses that varied between 12 and 24, as a 175
consequence of the reduction of MDT treatment from 24 to 12 doses as recommended by WHO, 176
(ii) the DNDS regimen only or (iii) the DNDR regimen and 24 doses of the MDT scheme. The 177
latter situation had occurred in a considerable number of cases (Table 1) and also in three of the 178
cases with drug associated mutation (Table 3). 179
Ampification and sequencing of rpoB, folP1, and gyrA; 180
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181
The results on amplification and DNA sequencing of part of the genes of folP1, rpoB, 182
and gyrA are presented in Table 2. A total of 92 samples (63.4%) yielded sequence results for at 183
least one gene fragment and informative sequences were obtained for 57 cases (61.9%) for folP1, 184
57 cases (61.9%) for rpoB and 77 cases (83.6%) for gyrA. Drug associated SNPs were detected 185
among three of the 57 samples for folP1 (5.3%), four of the 57 samples for rpoB (7%) and two of 186
the 77 samples for gyrA (2.6%). In addition, a statistically significant difference was observed 187
between BI and sequence results (Table 2). 188
Among the 23 of these biopsy samples that were tested for presence of PCR inhibitors, 21 189
had positive BI, one sample was BI=0 and from another sample we had no information on BI. 190
Among these samples, eight (35%) showed a PCR signal similar to that of the positive controls, 191
nine (39%) give weaker signals and six samples (26%) gave no PCR product at all (data not 192
shown), meaning that 65% of this sample selection showed some level of PCR inhibition for 193
gyrA (data not shown). We did not test PCR inhibition in the PCR systems for rpoB and folP1. 194
Regarding the nature of the SNPs, the three changes in folP1 were always a transition 195
from CCC to CGC in codon 55 (Pro to Arg); in the case of rpoB, all occurred at codon 531, two 196
presenting change from TCG to ATG (Ser to Met), one TCG to TTC (Ser to Phe) and one TCG 197
to TTG (Ser to Leu); the two cases with mutations in gyrA presented a transition from GCA to 198
GTA (Ala to Val) in codon 91. When considering this on the patient level, mutations suggestive 199
for drug resistant strains was observed in four cases, including one patient with a mutation in 200
rpoB only suggesting mono-resistance to rifampin, one case with SNPs in both folP1 and rpoB, 201
suggestive for multiple drug resistance (MDR) for rifampin and dapsone and two cases that were 202
mutated in folP1, rpoB and gyrA, strongly suggestive for MDR against the three main anti-203
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leprosy drugs (Table 2). The sequence results obtained with the four cases that presented SNPs at 204
Fiocruz were confirmed by Dr. Matsuoka in the Leprosy Research Center National Institute of 205
Infectious Diseases, Japan. 206
In addition to the drug resistant associated SNP in gyrA, this gene fragment also 207
presented a synonymous SNP in position 297 and as demonstrated in Table 2, among the 77 208
samples that were sequenced, 57 (74.03%) presented the C allele while 20 samples (25.97%) had 209
the T allele. The four cases with drug resistant associated SNPs presented the C allele. 210
Characteristics of patients with mutated strains. 211
Table 3 summarizes the data from DNA sequencing and mutations found in the four 212
patients, three being MDR. The first three cases were residents in ex-colonies for leprosy patients 213
in the Amazon region and all were submitted to the before-mentioned DNDS regimen in their 214
first disease episode. Case one, from the state of Para, presented the most characteristic 215
resistance features as his treatment failed in a second treatment course (first DNDS/Brazil and 216
then two courses of MDT/WHO). His last treatment course ended in 2007 while he presented 217
active LL disease in the beginning of 2008. The two cases from the Amazon State had also been 218
submitted to two complete treatment schemes before diagnosis of relapse, and their clinical 219
feature provoked the suspicion of drug resistance (DR). The first of these two patients has a 220
mutation on gyrA and we discovered that at the end of the second scheme (MDT/WHO), this 221
patient also had received ofloxacin but not according to a standard treatment scheme. Finally, the 222
fourth and quite intriguing case from the state of Espírito Santo, South-East Brazil, was 223
diagnosed with BL during first disease, presenting positive BI, but was negative in the second 224
disease course, seven years later, and classified as suffering from the BT form. Although this 225
patient presented a resistance-associated SNP in gyrA, we found no history of treatment with 226
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ofloxacin and eventual reinfection by an ofloxacin resistant strain from his relatives could be 227
possible. Among the 145 patients, 31% informed that they had relatives that were diagnosed for 228
leprosy, within five years before relapse diagnosis (Table 1 and 3). 229
230
DISCUSSION 231
The efficiency of the WHO MDT scheme for leprosy treatment is supported by the 232
dramatic decreases in disease prevalence and the low relapse rates in a short and medium time 233
frame. Therefore, relapse has not been considered a problem and organization of studies on this 234
disease characteristic was somewhat neglected, leading to the recent WHO initiative to organize 235
a resistance surveillance project in relapse cases, 26 years after having started MDT. This was 236
possible due to the development and standardization of molecular genotyping procedures of 237
genes associated with drug resistance (5, 16, 23, 24, 38, 39). 238
After the introduction of relapse surveillance by the WHO, many of the endemic 239
countries reported leprosy relapses. In addition, evaluation of the contribution of drug resistance 240
under an international network has been implemented, focusing on MB relapse cases (36). For 241
good quality data on relapse rates, in addition to laboratorial technology, uniformity of clinical 242
criteria relapse diagnosis is important and needs to be standardized within and among countries. 243
Although it not so difficult to diagnose leprosy relapse during the late MB disease form, 244
recognition of relapse is not so easy during early disease, especially in the Borderline spectrum 245
cases of disease and under field conditions (19, 20). 246
In the present study, 29.7% of the relapses were PB cases, 88.3% of these being BT, and 247
this after clinical examination by experienced leprologists and histopathological confirmation by 248
three different pathologists. This was also the case for the BT patient that presented mutations in 249
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genes rpoB, folP and gyrA and possibly, this patient, although being MDR, presented this disease 250
form because he (i) was diagnosed very soon after developing relapse, (ii) had a better immune 251
host defense response or (iii) had a different strain causing relapse, as observed in a considerable 252
number of relapse cases in another study, either by re-infection or strain selection (8). On the 253
other hand, selection of a particular part of the M. leprae population that caused first disease, for 254
being responsible for relapse is in accordance with Toman in 1981 (35) and Colston et al in 1987 255
(7), raising the possibility that “persistent” M leprae could cause relapse in a large proportion of 256
patients, the persistent bacilli presenting a metabolic state that resists the drug without the 257
presence of drug-associated mutations, also suggested by Pattyn (28) and Balagon et al (1). 258
Suspicion of DR or MDR in leprosy is raised mainly because of maintenance of clinical 259
symptoms, with or without evaluation of the presence of bacilli in skin smears and confirmation 260
by growth in the footpad of mice fed with antibiotics. Because bacteriological analysis by smear 261
microscopy is however not always reliable and advances in the elucidation of molecular events 262
responsible for drug resistance in mycobacteria have allowed the development of alternative 263
tools for drug-resistance screening (6). However, due to the need of technical expertise and 264
specialized equipment, this technique is executed in a limited number of centers in Brazil (38). 265
Nonetheless, SNP detection seems to be a more sensitive and is certainly much quicker for 266
detecting DR that the mouse footpad based technique (23, 33). In two very recent studies, 267
sequence analysis for DR in Latin American leprosy patients was reported, the first presenting 268
two cases with SNPs in rpoB and one case in gyrA, suggestive for resistance against respectively 269
rifampicin and ofloxacin, among 38 Mexican cases, suggesting the possible reemergence of DR 270
leprosy in a country where leprosy was considered eliminated (22). The second study included 271
230 mostly new leprosy cases, two being from Uruguay, 10 from Bolivia, 23 from Brazil and 272
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197 from Venezuela. Only two relapse cases presented SNPs in the three genes studied, one 273
from Venezuela in folP1 and one from Brazil folP1 and rpoB (34). 274
The mutations observed presently all have been reported in studies in other countries, 275
including the changes in codon 531 of rpoB causing a amino acid change from Ser to either Met 276
(n=1), Phe (n=2) or Leu (n=1). The SNP observed in folP1 in codon 55 (n=3), causing the 277
change of Pro to Arg and the mutation at codon 91 of gyrA (n=2) leading to change from Ala to 278
Val. These SNPs had earlier been described in several reports, including Honoré and Cole (17), 279
Williams et al. (37), Cambau et al. (6) and Gillis and Williams (14). In addition to the non-280
synonymous SNP in gyrA, we observed the allele distribution in the relapse cases of a recently 281
observed synonymous SNP at position 297 of gyrA (8, 25), showing that 74% of the cases 282
carried M. leprae of the SNP type gyrAC at position 297. Our own previous data (8) and the 283
recently published data from Singh et al (34) showed the correlation of the synonymous SNP 284
gyrA297T type with the SNP type 3 and SNP gyrAC with type 1 or 4 defined by Monot et al. 285
(25). Previous data showed the higher frequency of the SNP3 type in South-East Brazil (13) and 286
Latin-America (34) and the prevalence of the SNP gyrAC could be due to sampling from other 287
regions of Brazil. 288
We did not obtain PCR product and good quality sequences from all samples and this is 289
partly due to the inclusion of samples with low or zero bacterial counts and to the presence of 290
PCR-inhibitors, as evidenced by the reconstitution experiment. Indeed the presence of PCR 291
inhibitors in skin biopsy samples has been described before (32). 292
The significant difference between the period of time between first disease and relapse 293
between resistant and non-resistant cases is in agreement with Pattyn et al. (28), suggesting 294
difference of incubation period in these two kinds of relapses. One MDR relapse case however 295
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showed such a short incubation period (one month) that we suspect that this patient did not really 296
cured from his second disease course (Table 3). Our observation that all resistant cases were 297
males is in agreement with other studies (29, 30) and could be associated with the higher 298
prevalence of males in MB leprosy and more frequent irregular of self-administered drug intake 299
(including quinolosnes) in males, causing mainly secondary resistance. This is supported by the 300
recent observation of Singh et al. (34), showing the absence of primary drug resistance as 301
demonstrated by the lack of drug related mutations in strains from new leprosy patients. Indeed, 302
three out of four of the DR patients are from leprosy colonies that had received previous 303
Brazilian treatment regimen before MDT/WHO. Possibly, these cases, despite of receiving 304
regular monthly doses of the MDT/WHO scheme, might have been non compliant regarding the 305
daily self-administrated dose of combined dapsone and clofazimine. 306
Although DR does not seem to a problem in Brazil, one should note that the three older 307
DR cases had typical skin lesions of leprosy and good access to a health unit, and yet suffered 308
from late diagnostics, strongly suggesting the need of inclusion of ex-colony areas as “loci” for 309
epidemiological surveillance for relapse, as per norms defined by the Ministry of Health (4). 310
Also, the observation of two cases of MDR strains of M. leprae against the three most common 311
drugs for treatment is preoccupying and could become a serious threat for leprosy control. In 312
order to comply with the Global Surveillance of DR in Leprosy, it had been recommended to (i) 313
provide a technical guideline from the National Hansen’s Disease (Leprosy) Control Programme 314
(4) for establishment of relapse surveillance measures, (ii) include the study of drug resistance, 315
(iii) provide recommendations for the management of suspected relapse cases and (iv) design a 316
specific investigation form for the cases notified as relapse in the SINAN national information 317
system (3). In addition, we suggest the implementation within the leprosy control program, the 318
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monitoring of DR and MDR patients and their close contacts and to organize a reference frame 319
work 320
Our data show that development of DR isolates of M. leprae is contributing to leprosy 321
relapse in Brazil but that alternative causes are (i) bacterial persistence, (ii) immunosuppression 322
of the host, (iii) pregnancy, (iv) suffering from advanced leprosy, (v) re-infection and (vi) factors 323
associated with failures in operational health care weaknesses such as late diagnosis, inadequate 324
or irregular treatment of the disease and misclassification of earlier disease (8, 18, 19, 20). We 325
admit however that a limitation of this study is having used PCR sequencing for SNP detection, 326
with limitations regarding the detection of eventual minor mutant populations. In addition, 327
mutations outside of the part of the genes that was sequenced could have been missed. 328
329
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ACKNOWLEDGEMENTS 331
This study was possible through funds provided by CNPq/DECIT/MS-Brazil, grant 332
N401296/2005-9 and infrastructure of Laboratory of Molecular Biology Applied to 333
Mycobacteria and Leprosy Laboratory of FIOCRUZ, Rio de Janeiro, RJ, UFRJ coordination and 334
the collaboration of many health professionals from all six outpatient reference health units’ 335
participants, from five states of Brazil. 336
337
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447
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Table 1- Characteristics of relapse patients 448 449 Variables N=145 % Std.
Dev
1-Sex
Male 105 72.4
Female 40 27.6
2-Clinical Form
MB 102 70.3
PB 43* 29.7
3-Treatment regimen of first
disease course
MDT MB 12*
MDT MB 24
MDT PB
22
57
31
15.1
29.3
21.3
ROM** 2 1.3
DNDS +MDT 24 7 4.8
DNDS 14 9.6
Substitutive Regiment*** 12 8.2
4-Close Contact
with leprosy case
38⁄120 31.6
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5-Age (median value in years)
47.5 10.5
6- Time from cure to relapse
(median value in years)
All cases
DR-MDR cases
9.45
3.26
4.95
2.62
7-BaciIlary Index
(median value)
2.85
1.87
450 Notes: *Multidrug therapy with the number of doses between 12 to 24; **ROM: Rifampicin + 451
Ofloxacin + Minocicline; ***Replacement of Rifampicin by Ofloxacin, of Dapsone by 452
Clofazimine or combined use of Rifampicin and Clofazimine without Dapsone). Statistical 453
analysis was performed using the Fisher’s exact test. 454
455
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Table 2 – Results of DNA sequencing and mutations in folP1, rpoB and gyrA genes of M. leprae. 457
458
Genes Conclusive sequencing SNP present no SNP
IBCAT 0 1 2 P value
folP1 4/60 (6.6%) 22/41
(53.7%)
31/44
(70.5%)
<0.001 3 (5.3%) 54 (94.7%)
rpoB 5/60 (8.2%) 19/41
(46.3%)
33/44
(75.0%)
<0.001 4 (7%) 53 (93%)
gyrA 18/60
(29.5%)
27/41
(65.9%)
32/44
(72.7%)
<0.001 2 (2.6%) 75 (97.4%)
Total number of relapsed cases: 145
459
IBCAT: categorized bacillary index: 0, IB=0; 1, 0<IB< 3+ or 2, IB>3+; p value was of the three groups as calculated with the Fisher’s 460
exact test; SNPs are drug-resistant related only 461
462
463
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Table 3 – Summary of drug resistant relapse cases. 464
Results of DNA sequencing
Cases Sex Age CF BI His Treatment IP folP1 rpoB gyrA
1- PA M 49 LL 5 LL DNDS-MDT 24 1 mo folP1 55: CCC-CGC (Pro – Arg) rpoB 531: TCG-ATG (Ser – Met) No mutation
2- AM M 63 LL 4,5 LL DNDS-MDT 24 3.2 yr folP1 55: CCC-CGC (Pro – Arg) rpoB 531: TCG-TTC (Ser – Phe) gyrA91: GCA-GTA (Ala – Val)
3- AM M 46 BL 3,5 LL MDT 24 3.3 yr No mutation rpoB 531: TCG-TTG (Ser – Leu) No mutation
4- ES M 38 BT 6,6 BT MDT 12 6.6 yr folP1 55: CCC-CGC (Pro – Arg) rpoB 531: TCG-TTC (Ser – Phe) gyrA91: GCA-GTA (Ala – Val)
465
CF: clinical form; BI: Baciloscopic índex; His Histopathologic diagnostis; IP: incubation period of relapse: mo: month; yr: years; Pro: Proline; Arg: Arginine; 466
Ser: Serine; Phe: Phenylalanine; Leu: leucine; Met: Methionine; Ala: Alanine; Val: Valine; MDT/MB 24 (WHO): Rifampicin (RMP) 600 mg/month + 467
Clofazimine (CLZ) 300 mg/month (supervised) + DDS 100 mg + CLZ 50 mg/day, during a period between 12 to 18 months; DNDS (Brazil)- Rifampicin (RMP) 468
600 mg/day, 90 days + Dapsone (DDS) 100 mg/day up to five years until AFB negative. Case one received three treatment courses 469
470
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