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Drug and multidrug resistance among Mycobacterium leprae isolates from Brazilian relapsed leprosy patients.

ABSTRACT: Skin biopsy samples from 145 relapse leprosy cases and from five different regions in Brazil were submitted for sequence analysis of part of the genes associated with Mycobacterium leprae drug resistance. Single nucleotide polymorphisms (SNPs) in these genes were observed in M. leprae from 4 out of 92 cases with positive amplification (4.3%) and included a case with a mutation in rpoB only, another sample with SNPs in both folP1 and rpoB, and two cases showing mutations in folP1, rpoB, and gyrA, suggesting the existence of multidrug resistance (MDR). The nature of the mutations was as reported in earlier studies, being CCC to CGC in codon 55 in folP (Pro to Arg), while in the case of rpoB, all mutations occurred at codon 531, with two being a transition of TCG to ATG (Ser to Met), one TCG to TTC (Ser to Phe), and one TCG to TTG (Ser to Leu). The two cases with mutations in gyrA changed from GCA to GTA (Ala to Val) in codon 91. The median time from cure to relapse diagnosis was 9.45 years but was significantly shorter in patients with mutations (3.26 years; P = 0.0038). More than 70% of the relapses were multibacillary, including three of the mutation-carrying cases; one MDR relapse patient was paucibacillary.

SUBMITTER: da Silva Rocha A

PROVIDER: S-EPMC3372169 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Drug and multidrug resistance among Mycobacterium leprae isolates from Brazilian relapsed leprosy patients.

da Silva Rocha Adalgiza A Cunha Maria das Graças Md Diniz Lucia Martins LM Salgado Claudio C Aires Maria Araci P MA Nery José Augusto JA Gallo Eugênia Novisck EN Miranda Alice A Magnanini Monica M F MM Matsuoka Masanori M Sarno Euzenir Nunes EN Suffys Philip Noel PN de Oliveira Maria Leide W ML

Journal of clinical microbiology 20120411 6

Skin biopsy samples from 145 relapse leprosy cases and from five different regions in Brazil were submitted for sequence analysis of part of the genes associated with Mycobacterium leprae drug resistance. Single nucleotide polymorphisms (SNPs) in these genes were observed in M. leprae from 4 out of 92 cases with positive amplification (4.3%) and included a case with a mutation in rpoB only, another sample with SNPs in both folP1 and rpoB, and two cases showing mutations in folP1, rpoB, and gyrA, ...[more]

PMID: 22495562

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Project description:BackgroundLeprosy reactions, reversal reactions/RR and erythema nodosum leprosum/ENL, can cause irreversible nerve damage, handicaps and deformities. The study of Mycobacterium leprae-specific serologic responses at diagnosis in the cohort of patients enrolled at the Clinical Trial for Uniform Multidrug Therapy Regimen for Leprosy Patients in Brazil/U-MDT/CT-BR is suitable to evaluate its prognostic value for the development of reactions.MethodologyIgM and IgG antibody responses to PGL-I, LID-1, ND-O-LID were evaluated by ELISA in 452 reaction-free leprosy patients at diagnosis, enrolled and monitored for the development of leprosy reactions during a total person-time of 780,930 person-days, i.e. 2139.5 person-years, with a maximum of 6.66 years follow-up time.Principal findingsAmong these patients, 36% (160/452) developed reactions during follow-up: 26% (119/452) RR and 10% (41/452) had ENL. At baseline higher anti-PGL-I, anti-LID-1 and anti-ND-O-LID seropositivity rates were seen in patients who developed ENL and RR compared to reaction-free patients (p<0.0001). Seroreactivity in reactional and reaction-free patients was stratified by bacilloscopic index/BI categories. Among BI negative patients, higher anti-PGL-I levels were seen in RR compared to reaction-free patients (p = 0.014). In patients with 0<BI<3, (36 RR, 36 reaction-free), higher antibody levels to PGL-I (p = 0.014) and to LID-1 (p = 0.035) were seen in RR while difference in anti-ND-O-LID positivity was borderline (p = 0.052). Patients with BI≥3 that developed ENL had higher levels of anti-LID-1 antibodies (p = 0.028) compared to reaction-free patients. Anti-PGL-I serology had a limited predictive value for RR according to receiver operating curve/ROC analyses (area-under-the-curve/AUC = 0.7). Anti LID-1 serology at baseline showed the best performance to predict ENL (AUC 0.85).ConclusionsOverall, detection of anti-PGL-I, anti-LID-1 and anti-ND-O-LID antibodies at diagnosis, showed low sensitivity and specificity for RR prediction, indicating low applicability of serological tests for RR prognosis. On the other hand, anti-LID-1 serology at diagnosis has shown prognostic value for ENL development in BI positive patients.Trial registrationClinicalTrials.gov NCT00669643.

Project description:BACKGROUND:Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS:Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE:Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.

Project description:BackgroundReal-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens.Methodology/principal findingsThe specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing.Conclusion/significanceTaken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

Dataset Information

Drug and multidrug resistance among Mycobacterium leprae isolates from Brazilian relapsed leprosy patients.

Publications

Drug and multidrug resistance among Mycobacterium leprae isolates from Brazilian relapsed leprosy patients.

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