Equal rates of drug resistance in leprosy cases with relapse and recurrent/chronic Type 2 reaction: time to revise the guidelines for drug-resistance testing in leprosy?

BACKGROUND: Leprosy relapse/recurrence is a serious concern particularly in a leprosy-endemic nation such as India. It is believed that bacilli persisting even after multidrug therapy can cause relapse; recently, however, drug resistance as a cause for recurrences and chronic erythema nodosum leprosum (ENL) has been speculated. AIM: To study drug-resistance patterns in cases of leprosy relapse and chronic/recurrent (c/r)ENL. METHODS: This cross-sectional study conducted over a period of 1 year included patients diagnosed as having leprosy relapse and those with c/rENL. Skin biopsy specimens were examined by conventional PCR for resistance testing for rifampicin, dapsone and ofloxacin, respectively targeting the rpoB, folP and gyrA genes of Mycobacterium leprae. RESULTS: In total, 61 patients (25 smear-negative) were included in the study. Of these, 37 were diagnosed as having leprosy relapse and 24 as having c/rENL. Drug resistance to at least one drug was identified in 10 cases (16.4%). Rates of drug resistance were 5.4% (2 of 37) for dapsone, 10.8% (4 of 37) for rifampicin and 2.7% (1 of 37) for ofloxacin among cases of relapse, whereas it was 12.5% (3 of 24) and 8.3% (2 of 24) for dapsone and rifampicin respectively among those with c/rENL. Multidrug resistance was seen in 3.3% patients (2 of 61). CONCLUSION: Drug-resistance rate among those with c/rENL was almost equalled that of relapse. Smear-negative leprosy relapse cases also had resistance to bactericidal drugs. These findings call for modifications in criteria for testing under leprosy drug-resistance surveillance and all cases of relapse and those with recalcitrant c/rENL should be tested.

Antimicrobial resistance in leprosy: results of the first prospective open survey conducted by a WHO surveillance network for the period 2009-15.

OBJECTIVES: Antimicrobial resistance (AMR) is a priority for surveillance in bacterial infections. For leprosy, AMR has not been assessed because Mycobacterium leprae does not grow in vitro. We aim to obtain AMR data using molecular detection of resistance genes and to conduct a prospective open survey of resistance to antileprosy drugs in countries where leprosy is endemic through a WHO surveillance network. METHODS: From 2009 to 2015, multi-bacillary leprosy cases at sentinel sites of 19 countries were studied for resistance to rifampicin, dapsone and ofloxacin by PCR sequencing of the drug-resistance-determining regions of the genes rpoB, folP1 and gyrA. RESULTS: Among 1932 (1143 relapse and 789 new) cases studied, 154 (8.0%) M. leprae strains were found with mutations conferring resistance showing 182 resistance traits (74 for rifampicin, 87 for dapsone and 21 for ofloxacin). Twenty cases showed rifampicin and dapsone resistance, four showed ofloxacin and dapsone resistance, but no cases were resistant to rifampicin and ofloxacin. Rifampicin resistance was observed among relapse (58/1143, 5.1%) and new (16/789, 2.0%) cases in 12 countries. India, Brazil and Colombia reported more than five rifampicin-resistant cases. CONCLUSIONS: This is the first study reporting global data on AMR in leprosy. Rifampicin resistance emerged, stressing the need for expansion of surveillance. This is also a call for vigilance on the global use of antimicrobial agents, because ofloxacin resistance probably developed in relation to the general intake of antibiotics for other infections as it is not part of the multidrug combination used to treat leprosy.

Cortisol and proinflammatory cytokine profiles in type 1 (reversal) reactions of leprosy.

PURPOSE: Cortisol levels in the circulation and at the sites of peripheral inflammation regulate type 1 (Reversal) reactions in leprosy akin to delayed type hypersensitivity reactions (DTH). In this study we determine the extent to which the differential mRNA expression of genes encoding cortisone-cortisol shuttle enzymes (11 ß hydroxysteriod dehydrogenase I & II (11 ß HSD I & II)), circulatory levels of proinflammatory cytokines (IL-6, IL-7, IP-10, IL-17F, IL-23, TNF-α, IL-1ß, PDGF BB and CRP) and cortisol are associated with development of type 1 reactions in leprosy. METHODS: Urine, blood and incisional skin biopsy samples from site of lesions were collected from 49 newly diagnosed untreated leprosy cases in T1R and 51 cases not in reaction (NR). mRNA expression levels of genes encoding 11 ß HSD I & II in skin biopsy samples were determined by realtime PCR. Cortisol levels from the lesional skin biopsies, serum and urine samples and serum proinflammatory cytokine levels were measured using ELISA. RESULTS: The mean expression ratios of 11 ß HSD I & II are significantly lower in leprosy cases with T1R when compared to the NR leprosy cases. Cortisol levels in lesional skin biopsies and in urine are significantly lower (p=0.001) in leprosy cases with T1R. Serum cytokine levels of IP-10, IL-17F, IL-IL-6 and TNF-α are significantly higher (p<0.05) in leprosy cases with T1R when compared the NR leprosy cases. CONCLUSION: Our study indicated an association of urinary and lesional skin cortisol levels with the manifestation of T1R in leprosy. IP-10, IL-17F, IL-6 and TNF-α can be potential prognostic serological markers and gene expression markers for early detection of type 1 reactions in leprosy.

Potential of a metabolic gene (accA3) of M. leprae as a marker for leprosy reactions.

Understanding the mechanism(s) of reactions in leprosy remains a challenging task for both clinicians and basic scientists. While there is some understanding of host processes associated with different type of lepra reactions, there is very little information about bacterial factors triggering these inflammatory processes. This study is continuation of our earlier research programme on leprosy genomics in which significant transcription of 11 genes was observed during active disease and these included accA3 gene. In present study, we have investigated the potential of this gene or its gene product as molecular and or immunological marker for studying the reactions. Using quantitative Real-Time RT-PCR significant higher expression (mean log2 ratio=3.39) of accA3 was observed in specimens from leprosy reaction cases compared with cases without reactions. in silico homology model of this protein was analyzed for hydrophilic and B-cell epitope regions. Peptides with maximum antigenecity were selected, cloned, expressed and used to study sero-reactivity across the disease spectrum by indirect ELISA. While sero-reactivity was observed in leprosy cases the antibody levels did not vary significantly between the patient/s of same clinical type with and without reaction thereby indicating the limitation of this approach for this purpose. Measurement of transcription of this gene has, thus, potential as a molecular marker for monitoring the reactions.

Development and evaluation of real-time RT-PCR assay for quantitative estimation of viable Mycobacterium leprae in clinical samples.

Detection of live organisms by molecular methods has special significance in leprosy where causative organism can not be cultivated in vitro. Such techniques would be especially important for monitoring the progress of the disease. While real-time RT- PCR technology will be appropriate for this purpose, there is very little experience of use of such tools in leprosy. This study describes the development of a quantitative RT-PCR targeting 16S rRNA based on primers used in a semi quantitative RT-PCR and its application on clinical samples including slit scraping and biopsies. RNA was extracted from biopsies from 3 lepromatous leprosy (LL) cases and standard curve was generated by plotting crossing over point against the dilutions of input RNA quantity (number of bacilli used for RNA extraction). Real-time RT-PCR was performed for quantitative detection of live M. leprae in 28 slit (13/28 smear positive) scrappings and 32 biopsies (22/32 smear positive). Number of viable bacteria as estimated by solid stained bacilli and real-time PCR correlated (no difference p>0.05). The test achieved a theoretical analytical sensitivity limit of up to single live bacillus even considering 11.3% efficiency of RNA preparation which was calculated by spiking of known number of leprosy bacilli in non leprosy skin biopsies (PCR negative). All smear positive cases were positive by this assay. This assay appears to be a promising tool for detection and quantification of viable bacilli in selected clinical situations and should be of use even in smear negative cases also.