Efficacy of fixed duration multidrug therapy for the treatment of multibacillary leprosy: A prospective observational study from Northern India

Plain Language Summary

In endemic regions of several countries, the prevalence of leprosy has not come down to the level of elimination. On the contrary, new cases are being detected in large numbers from most of these areas in the majority of the endemic countries. Clinically, it is frequently noted that in spite of completion of a full course of multidrug therapy for 12 months, the lesions remain active, especially in cases with high bacteriological indices. Considering the above, the present study aimed at finding out the viability of Mycobacterium leprae strains from multidrug therapy-treated leprosy cases to find out their role in the transmission of disease. A total of 78 samples were collected initially to build up data on the viability of Mycobacterium leprae during multidrug therapy; samples were also collected at six months and 12 months intervals. Slit skin smears were collected from bacillary index (BI) positive new multibacillary leprosy cases in RNA Later vials (Sigma Co, USA.). RNAs were extracted from all the samples and were used for real-time polymerase chain reaction (two-step reaction). In our analysis, we observed that patients after one year of multibacillary multidrug therapy showed good signals for 16S rRNA, hsp18 and esxA genes, indicating that cases released from treatment were still harbouring viable bacilli in their system. We also observed that in this cohort of 36 patients, the Ct values of some of these samples increased for hsp18 and esxA genes (genes known to correlate with the growth of Mycobacterium leprae) even after 12 months of multidrug therapy. Presently, the multidrug therapy regimen has been shortened from two years to one year for multibacillary cases. It has been shown that the majority of relapses occur in multibacillary patients with Bl > 3+. By the time the patient was recognized as relapse, he/she might have infected several household contacts and neighbours. Viable Mycobacterium leprae from patients who have been released from treatment might have acted as the source for transmission of disease in the community, thus affecting the success of the leprosy eradication programme. The main aim of fixed duration multidrug therapy is to cure leprosy patients and to interrupt the transmission of leprosy to the community. Our study showed the positivity of viable bacilli in many patients after the completion of 12 months of multidrug therapy. Thus, it strongly suggests that the treatment regimen has to be redesigned for highly bacillated cases.

Introduction

Leprosy is a chronic infectious disease caused by Mycobacterium leprae inflicting skin and peripheral nerves and if left untreated may lead to disabilities due to nerve damage.¹ Although administration of multidrug therapy has drastically brought down the prevalence rate to a state of elimination (<1 case per 10,000 population size), the rate/ratio of new case detection has not yet declined even though the figures of a WHO report show a decline in the detection on new cases since 2008.² Out of the total new cases of the global population of leprosy, India alone had the major share of ≈ 60% cases (1,14,451 new cases) on record during the year 2019.³ In the recent past, the National Leprosy Eradication Programme carried out an innovative leprosy case detection campaign, which resulted in the detection of 34,000 new cases in 2016 from high endemic districts of the country. These cases accounted for 25% of new cases detected by the national programme in a year.⁴ In 2018, India reported 436 relapse cases out of a total number of 3361 in the world. It has been noted that relapse cases are mostly arising from multibacillary patients with high bacillary index ranging between 3+ and 5+ and who have completed the full regimen of multidrug therapy. This population of high bacillary index cases who are designated as “released from treatment” in an endemic region should ideally be free from viable bacilli but there will always remain dormant viable bacilli called “persisters,” which could be responsible for the occurrence of relapse. Moreover, by the time a relapse case is identified either in a field situation at a public health centre or in a tertiary care hospital, the patient might have transmitted infection to the household and neighboring contacts.

It has been mentioned that 99.9% Mycobacterium leprae are killed by rifampicin in active lepromatous patients.⁵ A highly bacillated (BI = 5+ to 6+) leprosy case has been estimated to have a Mycobacterium leprae load of 10¹⁰ to 10¹² bacilli per gram of tissue.⁶ In such cases after a full course of multidrug therapy even after 99.9% bacillary killing, ≈10⁷ to 10⁹ per gram of tissue Mycobacterium leprae will remain viable and grow uninhibitedly because Mycobacterium leprae specific cell-mediated immunity does not get restored in such cases.⁷ It is known that most of the time in paucibacillary and lower BI multibacillary cases the remaining bacilli disappear progressively over several years due to the immune response of the individuals, which is why the relapse rate has been around 2% in most places. However, in highly bacillated cases, cellular immunity is negligible, and these 2% will be responsible for the transmission of the disease and might progress to reactional episodes, leading to disability. Further, even after completion of treatment with adequate doses of multidrug therapy, cases of drug resistance and multidrug resistance of Mycobacterium leprae are being reported from various countries.^8-13 Relapse might occur either due to inadequate therapy, or after completing a full course of chemotherapy, which may vary from 0.6 to 3% in paucibacillary and 0.02– 0.8% in multibacillary cases due to wrong classification and insufficient therapy of PB cases. Further, a study has shown a relapse rate of 1.97/100 persons in multibacillary patients after 12 months of multi-drug therapy.¹⁴ Due to the occurrence of relapse in fully treated cases, questions arise about the efficacy of multidrug therapy in killing Mycobacterium leprae and in fully curing leprosy. At present, there is no satisfactory scientific method available for monitoring the efficacy of treatment in leprosy patients who relapse. As is well known, leprosy can also cause permanent damage if the patient remains without complete treatment or full cure.

Considering the above, the present work aimed at studying the efficacy of fixed duration multidrug therapy for multibacillary using real-time reverse transcription-polymerase chain reaction technology along with parameters of histopathological evidence of disease activity in high bacillary index cases.

Methods

Results

In our study, we used a method of quantifying bacteria by mRNA-based quantification, using real-time Q PCR.

Histopathology results

Sections of the skin biopsies were examined by haematoxylin and eosin staining and modified Fite-Faraco technique for Mycobacterium leprae at the time of recruitment and after completion of chemotherapy according to their bacillary index [Figure 1]. We did histology of a few representative samples at six and 12 monthly intervals of multidrug therapy. All these cases showed diffuse dermal infiltration with foamy macrophages with multiple solid-staining acid-fast bacilli. We observed that these patients, who were showing a viable load of bacilli at the time of release from treatment after a full course of fixed duration multidrug therapy, were also showing cellular activity on histopathology which showed the presence of granulomas [Figure 1].

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Figure 1:

Histological findings as per treatment duration: (A) At the time of recruitment. Arrows showing active granuloma. (B) At the time of release from treatment at the 12th month (H & E stain × 400). Arrows showing foamy macrophages with acid fast bacilli and active granuloma in panel B

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Development of standard curve for absolute quantification by determination of copy number of viable Mycobacterium leprae

For enumeration of a standard–curve, we performed the conventional polymerase chain reaction with three primer sets specific for three genes namely, 16SrRNA, hsp18 and esxA followed by gel elution and 10-fold serial dilutions. Mean crossing over points of three reactions of each dilution was plotted against log copy numbers to generate standard curve for absolute quantification [Figure 2].

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Figure 2:

Preparation of a standard curve for absolute quantification of (A) 16srRNA qPCR, (B) esxA qPCR and (C) hsp18 qPCR. Standard curves were obtained by plotting log copy number of eluted deoxyribonucleic acid carrying the target genes, ranging from 10⁷ to single copy, against the Ct value of the respective dilution

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Amplification efficiency of the assay for 16srRNA, hsp18 and esxA genes were found to be 95.184% (Amplification factor = 1.952), 99.05% (Amplification factor = 1.99), 96.14 % (Amplification factor = 1.96), respectively.

Bacterial load in leprosy patients

Mycobacterium leprae genome data showed the presence of a single copy of 16SrRNA, hsp18 and esxA gene per chromosome. The copy number was calculated by absolute quantification in each sample. To avoid the variation in the results, the same concentration of ribonucleic acid and complementary deoxyribonucleic acid was used for absolute quantification for all the three genes in each sample.

In this study, we found a significant increase in copy numbers for all three gene sets, i.e., 16srRNA, hsp18 and esxA after the completion of multidrug therapy. The highest bacillary load in terms of maximum copy number was observed in cases with BI > 4+ at the time of recruitment for all three genes. There was consistent detection of viable load of bacilli during the course of treatment by 16S rRNA in 24 patients (66.6%), hsp18 gene in 22 patients (61.1%) and esxA in 20 patients (55.5%) [Figure 3].

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Figure 3:

Determination of viable load of bacilli before and after the completion of 12 months of multibacillary multidrug therapy. Total ribonucleic acid from 36 multibacillary leprosy patients was collected before the multidrug therapy treatment started and after 12 months of treatment completion. Copy numbers of 16SrRNA (A), hsp18 (B) and esxA (C) were calculated. The time point of the post-treatment stage (duration in months) at which the sample was collected from each patient is shown on the X-axis. Copy number of messenger ribonucleic acid before treatment and after multidrug therapy treatment is shown on the Y-axis

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Discussion

Multidrug therapy has proved its effectiveness by bringing about a significant change in the prevalence of leprosy, both at the global and national level, by reducing the caseloads to the extent that leprosy no longer continues to be a major disease of public health importance. In addition, it has also been proved to be safe as well as acceptable to the patients.

Presence of viable Mycobacterium leprae in skin lesions has been noted after completion of the WHO fixed duration multidrug therapy regimen both in paucibacillary,¹⁷ and multibacillary patients.^17-19 Jamil et al. have suggested that multibacillary cases with an initial average BI ≥ 4+ should receive at least four years of standard WHO multidrug therapy.²⁰ Jethva et al. noted that 10% of the patients may continue to harbor live bacilli even after completion of two years of regular therapy.²¹ Some reports claim that the higher the bacillary index or shorter the duration of therapy, the higher the risk of relapse.²² Rigorous annual follow up for at least three years and even more for paucibacillary cases and nine years and even more for multibacillary cases must be observed.²³

In our study, we targeted the 16srRNA, esxA and hsp18 genes, to determine the viability load in skin in patients at the time of their recruitment before introduction to multidrug therapy regimen (zero day), after six months and 12 months of multidrug therapy when released from treatment. In the recent past Davis et al.²⁴ used quantitative real-time polymerase chain reaction expression for several gene targets of Mycobacterium leprae and correlated these with experimental mouse foot pad growth in both immunocompetent and immunosuppressed (nu/nu) mice and noted that hsp18 and esxA were the most expressive genes which correlated with the growth of Mycobacterium leprae. In our study, we observed a rise in expression of all three targets 16SrRNA, hsp18 and esxA gene targets in 24 (66.6%), 22 (61.1%) and 20 (55.5%), respectively, indicating that viable Mycobacterium leprae having the capacity to grow in a susceptible host are still present at the time of completion of treatment.

It was reported by Lini et al. that hsp18 gene is involved in the survival of pathogens under stressful conditions, which may account for the prolonged stability of hsp18 ribonucleic acid even during the period of chemotherapy.²⁵ Earlier reports by Wakade & Shetty using the mouse foot pad system demonstrated the presence of viable bacilli in skin lesions of fully treated borderline tuberculoid patients.²⁶ These results show that the presence of hsp18 mRNA in cases may indicate the viability of the pathogen. These findings support our findings of high levels of hsp18 mRNA in many patients at time of release from treatment.

The present finding demonstrates that this method could be adopted for specifically monitoring highly bacillated cases, to determine the need for continued multidrug therapy for longer periods. In addition to 16SrRNA, we included two more genes hsp18 and esxA in our study to know the viable load of bacilli having capacity to grow in lepromatous leprosy patients at the time of their release from treatment. We observed that the copy number of bacilli was up to ten,⁷ even at the time of release from treatment, in leprosy patients for both genes encoding hsp18 and esxA. A few studies have reported that the 16S rRNA signal remains for prolonged time even after the death of Mycobacterium leprae; because of that we included other two genes hsp18 and esxA in our study for the assessment of viability [Figure 3]. Increased copy number of hsp18 supports the findings of previous studies, which highlights the hsp18 gene association with stressful conditions. In all patients, the copy numbers of all the genes decreased gradually except for the group of 11 patients who were harbouring the viable load of bacilli, with BI > 4+ at the time of release from treatment [Figure 3]. Mouse foot pad assay has been used routinely to check the viability of the pathogen²⁷ and it has been also shown that the prolonged presence of viable bacilli has the properties to resist antimicrobials. It has been recently shown that hsp18 of Mycobacterium leprae, a homologue in Mycobacterium tuberculosis expressed under stress conditions,²⁸ and ESAT-6 protein expressing gene, esxA correlate with the exponential growth of Mycobacterium leprae in mouse foot pad. In the follow up cases after one year, some patients remained positive with viable bacilli having significant Mycobacterium leprae copy numbers with respect to 16SrRNA, hsp18 and esxA genes. These results highlight and support previous reports, which explain the role of hsp18 in stressful conditions, which may be one of the reasons for prolonged stability of ribonucleic acid after one year of multi-drug therapy.

From the present study, it could be concluded that borderline lepromatous/lepromatous leprosy patients with a high bacillary index could relapse after fixed-dose multidrug therapy for 12 months due to the growth of the remaining viable Mycobacterium leprae in these patients. As the Mycobacterium leprae specific cell-mediated immunity does not get restored in spite of fixed-dose multidrug therapy up to 12 months , the viable bacilli which are remaining find a favourable environment for their growth, which is noticed in nu/nu immunocompromised mice.²⁴ Further, this incomplete therapy which is not destroying all bacterial cells and is responsible for relapse might also cause development of rifampicin resistant Mycobacterium leprae and appear as a secondary rifampicin-resistant case. Secondary resistance to rifampicin in multibacillary relapse cases has been reported across the globe.^29,30 The main aim of fixed duration multidrug therapy is to cure leprosy patients and to interrupt the transmission of leprosy to the community. Therefore, it is desirable to treat/follow up on such cases until no viable bacilli remain in the patient.

Our study showed positivity of viable bacilli in many patients after the completion of 12 months of multidrug therapy. Thus, it strongly suggests that the treatment regimen has to be redesigned for highly bacillated cases. Therefore, the present regimen of multidrug therapy which advocates a fixed duration of treatment of 12 months for all multibacillary cases, irrespective of bacterial index, needs to be thoroughly debated and reconsidered.