Resistance to anti-leprosy drugs in multi-bacillary leprosy patients: The need for transformative action

Introduction

Mycobacterium leprae (M. leprae), the causative agent of leprosy, remains a significant public health concern, particularly in regions with limited access to healthcare and sanitation. Despite the availability of effective treatment options, the emergence of drug resistance threatens to undermine progress in controlling this ancient disease. Factors contributing to this phenomenon include patient non-adherence to therapy, inadequate monitoring of treatment response, and the intrinsic ability of the pathogen to evolve mechanisms to evade the effects of antimicrobial agents.¹ Drug resistance in Mycobacterium leprae can be detected using phenotypic methods, such as the mouse footpad assay, which tests bacterial viability but is time-consuming.² Molecular methods, like polymerase chain reaction (PCR) and DNA sequencing, are faster and identify specific gene mutations associated with resistance.³ Currently, molecular methods to detect rifampicin, dapsone, and ofloxacin resistance are available.

Resistance of M. leprae to rifampicin is primarily due to mutations in the rpoB gene, encoding the β-subunit of RNA polymerase, which prevent rifampicin from binding effectively.⁴ Dapsone resistance is primarily attributed to mutations in the folP1 gene, which encodes the dihydropteroate synthase enzyme. These genetic changes can lead to decreased drug susceptibility and, ultimately, treatment failure.⁴ Resistance of M. leprae to ofloxacin arises from mutations in the gyrA gene, which encodes the A subunit of DNA gyrase, reducing the drug’s ability to inhibit DNA replication.⁴

The emergence of drug resistance in M. leprae has significant implications for the control and management of leprosy. Resistance to the key antimicrobial agents can lead to treatment failures, increased disease transmission, and a higher risk of severe complications.

The present study was conducted to identify rates of primary and secondary drug resistance to rifampicin, dapsone, and ofloxacin in a tertiary care institute in Uttarakhand.

Methods

This prospective, cross-sectional study was conducted at a tertiary care institute in Uttarakhand over 2 years from July 2022 to June 2024 after obtaining approval from the institutional ethics committee (letter no. HIMS/RC/2021/263). Patients diagnosed with leprosy, having a bacteriological index (BI) ≥2 and fulfilling one of the sub-category criteria mentioned below, were recruited.

• a)

  New cases: Treatment naïve patients, who had never received anti-leprosy treatment

• b)

  Retreatment after loss to follow-up: Patients who received anti-leprosy treatment but did not complete 12 months of multi-drug therapy (MDT) in the past.

• c)

  Relapse: Patients presenting with signs and symptoms of the disease not attributable to a reaction after completing 12 months of MDT in the past.

• d)

  Persistent positive morphological index (MI): Patients with a positive MI after receiving 12 months of MDT.

• e)

  Chronic erythema nodosum leprosum (ENL): Patients with ENL for at least 24 weeks or more during which they required treatment for ENL either continuously or where any treatment-free period has been ≤27 days.

• f)

  Recurrent ENL: Patients with a second/subsequent episode of ENL occurring ≥28 days after stopping treatment for ENL.

All recruited patients were subjected to slit skin smear (SSS) examination and skin biopsy for calculating BI and MI and for histopathological confirmation, respectively. Further, samples were sent for antimicrobial drug resistance testing in the same manner as taking skin smears for BI/MI examination using a disposable stainless-steel blade. Samples were collected from two different sites from the most prominent skin lesions or the ear. The stainless-steel blade containing the tissue scrapings was rinsed into a 1.5 mL centrifuge tube (screw type) pre-filled with 1 mL of 70% ethanol, making sure that the tissue scrapings were washed from the surface of the blade and were suspended in the solution. Samples were then transported to Stanley Browne Laboratory, The Leprosy Mission Trust India in Delhi on the same day through speed-post for the detection of drug-resistant strains of M. leprae. PCR-based gene amplification was done using primers according to the guidelines in WHO’s ‘Global Surveillance of Drug Resistance in Leprosy 2008’ for the detection of mutations in the rpoB, gyrA, and folP genes of M. leprae.⁵

Results

From July 2022 to June 2024, 124 cases were seen in the leprosy clinic of the dermatology department. Table 1 outlines the patient distribution across each subcategory and specifies the number of cases for which antimicrobial resistance (AMR) was submitted.

Resistance to either rifampicin, dapsone, or ofloxacin was detected in eight (17%) samples [Table 2]. Primary resistance in patients with no known previous exposure to anti-leprosy drugs was found in four (20%) samples. Two (10%) patients each were resistant to rifampicin and ofloxacin. Of the 19 patients with chronic/recurrent ENL, one each was resistant to rifampicin and dapsone. The patient who was lost to follow-up after receiving four MDT packs and had reported after 2 years was resistant to rifampicin. One patient with persistent positive MI had dapsone resistance. None of the two patients of relapse (these patients had presented with new lesions for > 2 years after being released from treatment) had any identifiable resistance to rifampicin, dapsone, or ofloxacin. All four patients with rifampicin resistance had the F439L mutation. One patient each with dapsone resistance had the P55L and P55S mutations. Patients with ofloxacin resistance had the A91V and G89C mutations.

Patients having rifampicin resistance were initiated on modified MDT (100 mg minocycline + 400 mg ofloxacin + 50 mg clofazimine daily for 6 months, followed by 400 mg ofloxacin + 50 mg clofazimine daily for 18 months). Those with dapsone and ofloxacin resistance were continued on standard MDT. MI became zero after 6 months of modified MDT in two patients with primary rifampicin resistance and one retreatment case. Additionally, the patient with chronic/recurrent ENL and rifampicin resistance demonstrated marked clinical improvement post-initiation of modified MDT.

Discussion

Of the 47 samples sent for AMR testing, M. leprae could be amplified in 43. Resistance to rifampicin, dapsone, or ofloxacin was detected in eight (17%) tested samples. These findings align with the trends observed in reported global and Indian studies but also show significant variations, particularly in the frequency and type of mutations associated with drug resistance [Table 3].^6-16

Primary resistance in leprosy refers to drug resistance detected in patients who have never been exposed to anti-leprosy medications. It is critical because it indicates the transmission of drug-resistant strains of Mycobacterium leprae, complicating treatment from the outset and potentially leading to higher rates of treatment failure. The multinational WHO surveillance (2009-2015) reported primary resistance rates of 8.2% for rifampicin, 6.7% for dapsone, and 5.0% for ofloxacin.⁶ In the present study, a slightly higher rates have been reported for primary drug resistance, with 10% new cases showing resistance to each rifampicin and ofloxacin. However, none of the patients had dapsone resistance. This elevated resistance may be indicative of regional differences in AMR patterns or could reflect evolving trends in drug resistance due to genetic mutations or treatment practices over time. A similar trend of higher resistance was observed in the study by Lavania et al. (2009-2016), where they reported a 14.4% resistance to rifampicin and 13.6% to dapsone in a multi-state Indian study.⁷ However, most cases in their study were of relapse, where a high rate of resistance is expected. The study by Ahuja et al. (2017-2018) from West Bengal, India, reported a 4.8% primary resistance to rifampicin, a rate much lower than the 10% observed in the current study.⁸ However, their observation of a 12.4% resistance to ofloxacin is somewhat comparable to the 10% ofloxacin resistance in the reported cohort. Both studies underscore the growing concern of fluoroquinolone resistance in the treatment of leprosy, potentially complicating second-line treatment strategies. Globally, the study by Marijke et al. (2017-2020) in Belgium and the Comoros found no resistance to rifampicin, dapsone, or ofloxacin, in contrast to the present study’s 17% overall resistance rate.⁹ The absence of resistance in Braet’s study suggests regional variations in AMR patterns, potentially influenced by the lower prevalence of leprosy in these regions compared to endemic areas like India. Similarly, Andrade et al. (2018-2020) in Brazil reported very low resistance rates with only 0.25% of cases resistant to rifampicin and 1.18% resistant to dapsone.¹⁰

In a study from Chandigarh, India, AMR testing was done in patients with relapse and chronic/recurrent ENL. Here, 8.3% cases of chronic/recurrent ENL had rifampicin resistance, and 12.5% had dapsone resistance. The researchers further proposed to conduct AMR testing for all chronic/recurrent ENL cases with an aim to find if resistance is the underlying cause.¹² This aligns with the findings of the reported study. About 10.5% of patients with chronic/recurrent ENL were resistant to one of the anti-leprosy drugs tested, suggesting that ENL may be a clinical scenario where AMR testing is especially warranted. In a previous report from our institute, 7 of 8 reported cases of resistance had c/r ENL.¹³ Muddebihal et al. (2020) from New Delhi reported a 33.3% rifampicin resistance rate in reactional cases of leprosy; this further supports the inclusion of AMR testing in reactional cases to optimise treatment outcomes.¹⁴ Chhabra et al. (2021-2023) from Chandigarh, India, found that their three patients with reported rifampicin resistance had persistent skin lesions after completion of MDT.¹⁵ In another study from Chhattisgarh (India), high rates of resistance to rifampicin were noted in patients having a persistent positive MI post-release from treatment.¹⁶ One patient in the current study with persistent positive MI had dapsone resistance. Post-treatment positive MI can thus predict AMR, reinforcing the recommendation to conduct skin smears before release from treatment.

A meta-analysis of 22 studies highlights significant regional variations in the specific mutations associated with resistance to rifampicin, dapsone, and ofloxacin in Mycobacterium leprae. Among rifampicin-resistant strains, the Ser-to-Leu codon substitution (notably S425L, S531L, and S456L) was the most frequent mutation globally.¹⁷ In contrast, our study identified all four rifampicin-resistant cases with the F439L mutation, which has been previously reported infrequently in the literature.⁷ These findings underscore the importance of localised data collection, as recent Indian studies [Table 4] reveal unique mutation patterns for resistance to rifampicin, dapsone, and ofloxacin, reflecting the need for region-specific resistance surveillance.^8,12,13,15 Further validation using animal model experiments is required to determine whether the F439L mutation leads to functional alterations or is simply a sequencing artifact.

Limitations

The study’s conclusions are constrained by the small sample size and its conduct at a single tertiary care center, which may limit the generalisability of the findings. Furthermore, not all samples could be successfully amplified for analysis.

Conclusion

In conclusion, AMR testing in leprosy is crucial to guide effective management and should be implemented extensively across all geographical regions to identify regional variations. Widespread availability of reference laboratories for PCR-based resistance detection is essential, not only for new and relapse cases but also for patients with recurrent or chronic ENL and persistent positive MI.