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

BACKGROUND: 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. Clinically, it is frequently noted that despite completion of multibacillary multidrug therapy for 12 months, the lesions remain active, especially in cases with high bacteriological indices. AIM: The present study focused on finding out the viable number of Mycobacterium leprae during the 12-month regimen of multibacillary multidrug therapy, at six and 12 months intervals and, attempting to determine their role in disease transmission. METHODS: Seventy eight cases of multibacillary leprosy cases were recruited from leprosy patients registered at The Leprosy Mission hospitals at Shahdara (Delhi), Naini (Uttar Pradesh) and Champa (Chhattisgarh), respectively. Slit skin smears were collected from these patients which were transported to the laboratory for further processing. Ribonucleic acid was extracted by TRIzol method. Total Ribonucleic acid was used for real-time reverse transcription-polymerase chain reaction (two-step reactions). A standard sample with a known copy number was run along with unknown samples for a reverse transcription-polymerase chain reaction. Patients were further assessed for their clinical and molecular parameters during 6th month and 12th month of therapy. RESULTS: All 78 new cases showed the presence of a viable load of bacilli at the time of recruitment, but we were able to follow up only on 36 of these patients for one year. Among these, using three different genes, 20/36 for esxA, 22/36 for hsp18 and 24/36 for 16S rRNA cases showed viability of M. leprae at the time of completion of 12 months of multidrug therapy treatment. All these positive patients were histopathologically active and had bacillary indexes ranging between 3+ and 4+. Patients with a high copy number of the Mycobacterium leprae gene, even after completion of treatment as per WHO recommended fixed-dose multidrug therapy, indicated the presence of live bacilli. LIMITATIONS: Follow up for one year was difficult, especially in Delhi because of the migratory nature of the population. Patients who defaulted for scheduled sampling were not included in the study. CONCLUSION: The presence of a viable load of bacilli even after completion of therapy may be one of the reasons for relapse and continued transmission of leprosy in the community.

Molecular detection of multidrug-resistant Mycobacterium leprae from Indian leprosy patients.

OBJECTIVES: The emergence of multidrug-resistant (MDR) organisms for any infectious disease is a public health concern. Global efforts to control leprosy by intensive chemotherapy have led to a significant decrease in the number of registered patients. Currently recommended control measures for treating leprosy with multidrug therapy (MDT) were designed to prevent the spread of dapsone-resistant Mycobacterium leprae strains. Here we report the identification of MDR M. leprae from relapse leprosy patients from endemic regions in India. METHODS: Resistance profiles to rifampicin, dapsone and ofloxacin of the isolated strains were confirmed by identification of mutations in genes previously shown to be associated with resistance to each drug. Between 2009-2016, slit-skin smear samples were collected from 239 relapse and 11 new leprosy cases from hospitals of The Leprosy Mission across India. DNA was extracted from the samples and was analysed by PCR targeting the rpoB, folP and gyrA genes associated with resistance to rifampicin, dapsone and ofloxacin, respectively, in M. leprae. M. leprae Thai-53 (wild-type) and Zensho-4 (MDR) were used as reference strains. RESULTS: Fifteen strains showed representative mutations in at least two resistance genes. Two strains showed mutations in all three genes responsible for drug resistance. Seven, seven and one strain, respectively, showed mutations in genes responsible for rifampicin and dapsone resistance, for dapsone and ofloxacin resistance and for rifampicin and ofloxacin resistance. CONCLUSION: This study showed the emergence of MDR M. leprae in MDT-treated leprosy patients from endemic regions of India.

Drug resistance patterns in Mycobacterium leprae isolates from relapsed leprosy patients attending The Leprosy Mission (TLM) Hospitals in India.

Implementation of multidrug therapy (MDT) in leprosy control programmes has significantly reduced the global prevalence of the disease in the last two decades. After many years of use of MDT, it is expected that drug resistance in Mycobacterium leprae may emerge. This is a major concern, especially during the stage of elimination. In the present study, slit-skin smears were collected from 140 leprosy relapse cases from different Leprosy Mission hospitals across India. DNA extracted from 111 (79%) of these samples was analysed for the genes associated with drug resistance in M. leprae. More than 90% of the patients relapsed as multibacillary (MB) cases. In our study, four (3.6%) of the DNA samples analysed showed mutations associated with rifampicin resistance. We also observed that mutations associated with resistance to dapsone and ofloxacin were observed in 9 (8.1%) of the DNA samples each; two samples had both dapsone and ofloxacin resistance. Further surveillance and appropriate interventions are needed to ensure the continued success of chemotherapy for leprosy.

Serological responses to prednisolone treatment in leprosy reactions: study of TNF-α, antibodies to phenolic glycolipid-1, lipoarabinomanan, ceramide and S100-B.

BACKGROUND: Corticosteroids have been extensively used in the treatment of immunological reactions and neuritis in leprosy. The present study evaluates the serological response to steroid treatment in leprosy reactions and neuritis. METHODS: Seven serological markers [TNF-α, antibodies to Phenolic glycolipid-1 (PGL-1 IgM and IgG), Lipoarabinomannan (LAM IgG1 and IgG3), C2-Ceramide and S100 B] were analyzed longitudinally in 72 leprosy patients before, during and after the reaction. At the onset of reaction these patients received a standard course of prednisolone. The levels of the above markers were measured by Enzyme linked immunosorbent assay (ELISA) and compared with the individuals own value in the month prior to the reaction and presented as percentage increase. RESULTS: One month before the reaction individuals showed a varying increase in the level of different markers such as TNF-α (53%) and antibodies to Ceramide (53%), followed by to PGL-1 (51%), S100B (50%) and LAM (26%). The increase was significantly associated with clinical finding of nerve pain, tenderness and new nerve function impairment. After one month prednisolone therapy, there was a fall in the levels [TNF-α (60%), C2-Ceramide (54%), S100B (67%), PGL-1(47%) and LAM (52%)] with each marker responding differently to steroid. CONCLUSION: Reactions in leprosy are inflammatory processes wherein a rise in set of serological markers can be detected a month before the clinical onset of reaction, some of which remain elevated during their action and steroid treatment induces a variable fall in the levels, and this forms the basis for a variable individual response to steroid therapy.

Comparing the clinical and histological diagnosis of leprosy and leprosy reactions in the INFIR cohort of Indian patients with multibacillary leprosy.

BACKGROUND: The ILEP Nerve Function Impairment in Reaction (INFIR) is a cohort study designed to identify predictors of reactions and nerve function impairment in leprosy. The aim was to study correlations between clinical and histological diagnosis of reactions. METHODOLOGY/PRINCIPAL FINDINGS: Three hundred and three newly diagnosed patients with World Health Organization multibacillary (MB) leprosy from two centres in India were enrolled in the study. Skin biopsies taken at enrolment were assessed using a standardised proforma to collect data on the histological diagnosis of leprosy, leprosy reactions and the certainty level of the diagnosis. The pathologist diagnosed definite or probable Type 1 Reactions (T1R) in 113 of 265 biopsies from patients at risk of developing reactions whereas clinicians diagnosed skin only reactions in 39 patients and 19 with skin and nerve involvement. Patients with Borderline Tuberculoid (BT) leprosy had a clinical diagnosis rate of reactions of 43% and a histological diagnosis rate of 61%; for patients with Borderline Lepromatous (BL) leprosy the clinical and histological diagnosis rates were 53.7% and 46.2% respectively. The sensitivity and specificity of clinical diagnosis for T1R was 53.1% and 61.9% for BT patients and 61.1% and 71.0% for BL patients. Erythema Nodosum Leprosum (ENL) was diagnosed clinically in two patients but histologically in 13 patients. The Ridley-Jopling classification of patients (n = 303) was 42.8% BT, 27.4% BL, 9.4% Lepromatous Leprosy (LL), 13.0% Indeterminate and 7.4% with non-specific inflammation. This data shows that MB classification is very heterogeneous and encompasses patients with no detectable bacteria and high immunological activity through to patients with high bacterial loads. CONCLUSIONS/SIGNIFICANCE: Leprosy reactions may be under-diagnosed by clinicians and increasing biopsy rates would help in the diagnosis of reactions. Future studies should look at sub-clinical T1R and ENL and whether they have impact on clinical outcomes.

Predicting neuropathy and reactions in leprosy at diagnosis and before incident events-results from the INFIR cohort study.

BACKGROUND: Leprosy is a disease of skin and peripheral nerves. The process of nerve injury occurs gradually through the course of the disease as well as acutely in association with reactions. The INFIR (ILEP Nerve Function Impairment and Reactions) Cohort was established to identify clinically relevant neurological and immunological predictors for nerve injury and reactions. METHODOLOGY/PRINCIPAL FINDINGS: The study, in two centres in India, recruited 188 new, previously untreated patients with multi-bacillary leprosy who had no recent nerve damage. These patients underwent a series of novel blood tests and nerve function testing including motor and sensory nerve conduction, warm and cold detection thresholds, vibrometry, dynamometry, monofilament sensory testing and voluntary muscle testing at diagnosis and at monthly follow up for the first year and every second month for the second year. During the 2 year follow up a total of 74 incident events were detected. Sub-clinical changes to nerve function at diagnosis and during follow-up predicted these new nerve events. Serological assays at baseline and immediately before an event were not predictive; however, change in TNF alpha before an event was a statistically significant predictor of that event. CONCLUSIONS/SIGNIFICANCE: These findings increase our understanding of the processes of nerve damage in leprosy showing that nerve function impairment is more widespread than previously appreciated. Any nerve involvement, including sub-clinical changes, is predictive of further nerve function impairment. These new factors could be used to identify patients at high risk of developing impairment and disability.

Voluntary muscle testing and dynamometry in diagnosis of motor impairment in leprosy: a comparative study within the INFIR Cohort Study.

AIM: To evaluate hand muscle weakness detected through dynamometry as an indicator for change in motor nerve function detected by Voluntary Muscle Testing (VMT) of ulnar and median nerves. DESIGN: The research was carried out as part of the INFIR Cohort Study among 303 subjects newly diagnosed with MB leprosy in two centres in UP state, northern India. METHODS: To assess grip strength, key pinch and pulp-to-pulp pinch we adapted the cuffs of adult and neonatal sphygmomanometers. The testing was carried out at diagnosis and at each visit during a 2-year follow-up. RESULTS: 303 subjects with newly diagnosed MB leprosy were included in the study. We found statistically significant differences in grip strength, key pinch and pulp-to-pulp pinch between groups defined by ulnar VMT grades at time of diagnosis. There was also a statistically significant difference in hand grip between groups defined by median VMT at diagnosis. In each case, strength tended to reduce with increasing motor involvement. We explored reduction in grip strength, key pinch or pulp-to-pulp pinch as indicators of change in ulnar VMT during follow-up. A 25% reduction over two visits was the most effective indicator. Changes were also associated with marginal changes in motor and sensory nerve function, most commonly associated with Type I reactions. CONCLUSION: Dynamometry is recommended as an additional method that may be used to monitor changes in nerve function in leprosy, particularly in subjects with early motor impairment of the ulnar nerve.

Early diagnosis of neuropathy in leprosy--comparing diagnostic tests in a large prospective study (the INFIR cohort study).

BACKGROUND: Leprosy is the most frequent treatable neuromuscular disease. Yet, every year, thousands of patients develop permanent peripheral nerve damage as a result of leprosy. Since early detection and treatment of neuropathy in leprosy has strong preventive potential, we conducted a cohort study to determine which test detects this neuropathy earliest. METHODS AND FINDINGS: One hundred and eighty-eight multibacillary (MB) leprosy patients were selected from a cohort of 303 and followed for 2 years after diagnosis. Nerve function was evaluated at each visit using nerve conduction (NC), quantitative thermal sensory testing and vibrometry, dynamometry, monofilament testing (MFT), and voluntary muscle testing (VMT). Study outcomes were sensory and motor impairment detected by MFT or VMT. Seventy-four of 188 patients (39%) had a reaction, neuritis, or new nerve function impairment (NFI) event during a 2-year follow-up. Sub-clinical neuropathy was extensive (20%-50%), even in patients who did not develop an outcome event. Sensory nerve action potential (SNAP) amplitudes, compound motor action potential (CMAP) velocities, and warm detection thresholds (WDT) were most frequently affected, with SNAP impairment frequencies ranging from 30% (median) to 69% (sural). Velocity was impaired in up to 43% of motor nerves. WDTs were more frequently affected than cold detection thresholds (29% versus 13%, ulnar nerve). Impairment of SNC and warm perception often preceded deterioration in MF or VMT scores by 12 weeks or more. CONCLUSIONS: A large proportion of leprosy patients have subclinical neuropathy that was not evident when only MFT and VMT were used. SNC was the most frequently and earliest affected test, closely followed by WDT. They are promising tests for improving early detection of neuropathy, as they often became abnormal 12 weeks or more before an abnormal monofilament test. Changes in MFT and VMT score mirrored changes in neurophysiology, confirming their validity as screening tests.

The INFIR Cohort Study: investigating prediction, detection and pathogenesis of neuropathy and reactions in leprosy. Methods and baseline results of a cohort of multibacillary leprosy patients in north India.

The aim of this study was to find predictors of neuropathy and reactions, determine the most sensitive methods for detecting peripheral neuropathy, study the pathogenesis of neuropathy and reactions and create a bank of specimen, backed up by detailed clinical documentation. A multi-centre cohort study of 303 multibacillary leprosy patients in Northern India was followed for 2 years. All newly registered MB patients requiring a full course of MDT, who were smear positive and/or had six or more skin lesions and/or had two or more nerve trunks involved, were eligible. A detailed history was taken and physical and neurological examinations were performed. Nerve function was assessed at each visit with nerve conduction testing, warm and cold detection thresholds, vibrometry, dynamometry, monofilaments and voluntary muscle testing. Because the latter two are widely used in leprosy clinics, they were used as 'gold standard' for sensory and motor impairment. Other outcome events were type 1 and 2 reactions and neuritis. All subjects had a skin biopsy at registration, repeated at the time of an outcome event, along with a nerve biopsy. These were examined using a variety of immunohistological techniques. Blood sampling for serological testing was done at every 4-weekly clinic visit. At diagnosis, 115 patients had an outcome event of recent onset. Many people had skin lesions overlying a major nerve trunk, which were shown to be significantly associated with an increased of sensory or motor impairment. The most important adjusted odds ratios for motor impairment were, facial 4.5 (1.3-16) and ulnar 3.5 (1.0-8.5); for sensory impairment they were, ulnar 2.9 (1.3-6.5), median 3.6 (1.1-12) and posterior tibial 4.0 (1.8-8.7). Nerve enlargement was found in 94% of patients, while only 24% and 3% had paraesthesia and nerve tenderness on palpation, respectively. These increased the risk of reactions only marginally. Seven subjects had abnormal tendon reflexes and seven abnormal joint position sense. In all but one case, these impairments were accompanied by abnormalities in two or more other nerve function tests and thus seemed to indicate more severe neuropathy. At diagnosis, 38% of a cohort of newly diagnosed MB leprosy patients had recent or new reactions or nerve damage at the time of intake into the study. The main risk factor for neuropathy found in this baseline analysis was the presence of skin lesions overlying nerve trunks. They increased the risk of sensory or motor impairment in the concerned nerve by 3-4 times. For some nerves, reactional signs in the lesions further increased this risk to 6-8 times the risk of those without such lesions. Patients with skin lesions overlying peripheral nerve trunks should be carefully monitored for development of sensory or motor impairment.

The INFIR Cohort Study: assessment of sensory and motor neuropathy in leprosy at baseline.

AIM: To compare different method(s) to detect peripheral neuropathy in leprosy and to study the validity of the monofilament test (MF) and the voluntary muscle test (VMT) as standard tests of nerve function. DESIGN: A multi-centre cohort study of 303 multibacillary (MB) leprosy patients. METHODS: Newly registered MB patients requiring a full course of MDT were recruited in two leprosy outpatient clinics in North India. Controls were people without leprosy or neurological conditions, attending the dermatological outpatient departments of the same clinics. Nerve function was evaluated electrophysiologically using standard parameters for sensory and motor nerve conduction (NC) testing, warm and cold detection thresholds (W/CDT), vibration perception thresholds, dynamometry, MF and VMT. The latter two defined the outcomes of sensory and motor impairment. RESULTS: 115 patients had nerve damage or a reaction of recent onset at diagnosis. Sensory and motor amplitudes and WDTs were the most frequently abnormal. Among the nerves tested, the sural and posterior tibial were the most frequently impaired. In the ulnar nerve, sensory latencies were abnormal in 25% of subjects; amplitudes in 40%. Ulnar above-elbow motor conduction velocities were abnormal in 39% and amplitudes 32%. WDTs were much more frequently affected than CDTs in all nerves tested. The thresholds of all test parameters differed significantly between controls and patients, while only some differed between patients with and without reaction. Good concordance was observed between MF results and sensory latencies and velocities (direct concordance 80% for the ulnar). However, a proportion of nerves with abnormal MF results tested normal on one or more of the other tests or vice versa. Concordance between VMT and motor conduction velocities was good for the ulnar nerve, but for the median and peroneal nerves, the proportion impaired by VMT out of those with abnormal motor conduction was very low. CONCLUSIONS: Concordance between monofilaments and other sensory function test results was good, supporting the validity of the monofilaments as standard screening test of sensory function. Concordance between VMT results and motor nerve conduction was good for the ulnar nerve, but very few median and peroneal nerves with abnormal conduction had an abnormal VMT. A more sensitive manual motor test may be needed for these nerves. Of the nerve assessment tests conducted, NC amplitudes and warm sensation were the most frequently affected. Therefore, nerve conduction studies and WDT measurements appear to be most promising tests for early detection of leprous neuropathy. The pattern of concordance between tactile and thermal sensory impairment failed to support the hypothesis that small fibre neuropathy always precedes large fibre damage. Warm sensation was more frequently affected than cold sensation. This could indicate that unmyelinated C fibres are more frequently affected than small myelinated Asigma fibres.