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.

A Novel Therapeutic and Prophylactic Vaccine against Tuberculosis Using the Cynomolgus Monkey Model and Mouse Model.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65 + IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse model compared to the BCG. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality. The BCG prime and HSP65 + IL-12/HVJ vaccine (boost) by the prime-boost method showed a synergistic prophylactic effect in the monkey. Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys.HVJ-Envelope/HSP65 DNA + IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the ESR, and augmented the immuneresponses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials.

Polymorphisms in Toll-like receptor 4 (TLR4) are associated with protection against leprosy.

Accumulating evidence suggests that polymorphisms in Toll-like receptors (TLRs) influence the pathogenesis of mycobacterial infections, including leprosy, a disease whose manifestations depend on host immune responses. Polymorphisms in TLR2 are associated with an increased risk of reversal reaction, but not susceptibility to leprosy itself. We examined whether polymorphisms in TLR4 are associated with susceptibility to leprosy in a cohort of 441 Ethiopian leprosy patients and 197 healthy controls. We found that two single nucleotide polymorphisms (SNPs) in TLR4 (896G>A [D299G] and 1196C>T [T399I]) were associated with a protective effect against the disease. The 896GG, GA and AA genotypes were found in 91.7, 7.8 and 0.5% of leprosy cases versus 79.9, 19.1 and 1.0% of controls, respectively (odds ratio [OR] = 0.34, 95% confidence interval [CI] 0.20-0.57, P < 0.001, additive model). Similarly, the 1196CC, CT and TT genotypes were found in 98.1, 1.9 and 0% of leprosy cases versus 91.8, 7.7 and 0.5% of controls, respectively (OR = 0.16, 95% CI 0.06--.40, P < 0.001, dominant model). We found that Mycobacterium leprae stimulation of monocytes partially inhibited their subsequent response to lipopolysaccharide (LPS) stimulation. Our data suggest that TLR4 polymorphisms are associated with susceptibility to leprosy and that this effect may be mediated at the cellular level by the modulation of TLR4 signalling by M. leprae.

Sensory testing in leprosy: comparison of ballpoint pen and monofilaments.

The 10 g monofilament has been replaced by the ballpoint pen in routine sensory testing of nerves in leprosy control in Ethiopia. Results of sensory testing between the ballpoint pen and different monofilaments on hands and feet were compared. Ballpoint pen underdiagnosis of loss of sensation was defined to occur when the pen was felt and the monofilament was not. Differences were evaluated both for individual test points (test point level) and for the test points of extremities collectively (extremity level). An extremity (either a hand or a foot) was defined as having sensory nerve function impairment (SNFI) if a supplying nerve had SNFI, which was the case when sensation was absent in two or more test points in the area supplied by that nerve. At test point level, the percentages with ballpoint pen underdiagnosis relative to the 2, 10, 20 and 50 g monofilaments were 40, 21, 9 and 7%, respectively, in the hands, and 47, 30, 15 and 7% in the feet. Ballpoint pen underdiagnosis percentages of SNFI at extremity level were 32, 18, 8 and 9% in the hands, and 37, 26, 14 and 6% in the feet. The risk of ballpoint pen underdiagnosis appears to be higher in extremities without visible damage. In conclusion, substantial levels of underdiagnosis of sensory loss with the ballpoint pen were observed. However, the consequences for the prognosis of treatment with corticosteroids in patients with the more subtle sensation loss noted here need to be established. Development and testing of guidelines is a prerequisite for the use of the ballpoint pen.

Social stigma: a comparative qualitative study of integrated and vertical care approaches to leprosy.

Integration of leprosy into the general health system is very much emphasized by health care planners. One prime reason stated for this is to reduce stigma attached to this disease. This study was conducted in the state of Maharashtra, India, to compare the level of social stigma towards leprosy in communities with a vertical and an integrated programme. The data were collected in three areas of five villages each. The first two areas were in an integrated programme to test for internal consistency and the third in a vertical programme. All the leprosy patients with visible deformities in these villages were enrolled in the study, and an in-depth stigma measurement scale was administered. In addition, focus group discussions were conducted among the family members of leprosy patients and participative rural appraisal was done in the communities. The data were analysed using qualitative methods. A total of 24 leprosy patients with visible deformities participated in the in-depth stigma measurement exercise from 15 villages. Fifteen focus group discussions were conducted with families of leprosy patients and an equal number of participatory rural appraisals with communities were done. The results show that social stigma was virtually non-existent among the communities with the integrated approach and minimally experienced by leprosy patients in this model. However, a high level of self-stigmatization among leprosy patients was observed in the vertical approach and equally a high level of social stigma was found in their communities, which led to reduced interaction between the leprosy patients and their communities. The integrated approach to community-based primary health care is effective in reducing leprosy stigma in society.

Can people affected by leprosy at risk of developing plantar ulcers be identified? A field study from central Ethiopia.

In the ALERT leprosy control programme, 75 people affected by leprosy, in three different geographical areas, were investigated. Each person was documented as having anaesthesia to the 10 g monofilament. The study sought to determine why some people developed ulcers whilst others did not. According to the records, 43 had an ulcer during the last 5 years but 32 had never had an ulcer. In order to examine protective sensation on the sole of the foot, various sensory modalities were tested and the functional anatomy of the foot was examined. The results showed, as may be expected, that it is not possible to define a specific threshold for protective sensation that could be applied to all cases. Some people with only slightly diminished sensation developed ulcers, while many others with almost complete anaesthesia remained ulcer-free. In these rural communities, being a farmer reduced the risk of developing an ulcer, but no other demographic features were significant. Graded monofilaments were found to be the most appropriate test, with loss of sensation at any of five points tested being a 'positive' result. The 10 g filament was the most sensitive, but only 43% of feet identified by this test actually developed an ulcer. As people with partial loss of sensation were excluded from this study, this figure may be lower under programme conditions. The 50 g and 100 g filaments decrease the number of feet identified as at risk, but increase the percentage which actually develop an ulcer, to 46% and 49%, respectively. An appropriate test for selecting those for special programmes which may have a limited capacity, for example the provision of subsidized footwear or involvement in self-care groups, would be a 100 g filament, which would detect 86% of those feet likely to develop an ulcer, while reducing the number of those selected who are not at great risk. Vibrometry was found to be no better than graded filaments and an examination of functional anatomy did not help in identifying those at risk.

Dynamics of impairment during and after treatment: the AMFES cohort.

This study investigates the dynamics of impairment during and after multidrug therapy treatment for the patient cohort of the prospective ALERT MDT Field Evaluation Study (AMFES). The impairment status was compared at intake, at release from treatment (rft), and at the time of the latest survey between 24 and 48 months after release from treatment (follow-up). The eye-hand-foot impairment score (EHF score), which is the sum of the WHO impairment grades of the eyes, hands, and feet, was used as tool for comparison. In all, 433 out of the 592 patients (224 PB and 209 MB) completed treatment in time and were assessed at release from treatment. The risk of getting impaired was 4% for the 113 PB and 21% for the 91 MB patients who were initially free from impairment. Out of the 111 initially impaired PB patients, 41% recovered or improved and 13% worsened in EHF score. For the 118 initially impaired MB patients, these figures were: recovery or improvement 43% and worsening 13%. Three hundred and twenty-three out of the 433 patients (158 PB and 165 MB) had a follow-up examination in between the next 24-48 months after rft. The risks of impairment at follow-up were 6% for the 79 PB and 18% for the 77 MB patients without impairment at rft. Out of the 79 PB patients with impairment at rft, 35% recovered or improved and 28% worsened. For the 88 impaired MB patients, these figures were: recovery or improvement 26% and worsening 27%. Patients showed a tendency to compensate EHF score improvement before rft by worsening after rft and vice versa. The first main conclusion is that the impairment status at intake was by far the most important determinant for future impairment. The second one is that the dynamics of impairment were less favourable after rft than before. Little is known about the long-term fate of leprosy patients with irreversible nerve damage and the associated risk of developing severe secondary impairment. Especially in this era of the leprosy elimination goal, we should give this accumulating patient group due attention in research and health policy agendas.

The ALERT MDT Field Evaluation Study (AMFES): a descriptive study of leprosy in Ethiopia. Patients, methods and baseline characteristics.

The ALERT MDT Field Evaluation Study (AMFES) is a long-term prospective study of 650 patients (594 new cases and 56 relapses after dapsone monotherapy), treated with fixed-duration multiple-drug therapy (MDT), as recommended by WHO. Follow-up has continued for up to 11 years from the start of treatment. This paper presents the methodology of the study and the baseline characteristics of the cohort, while accompanying papers examine the incidence of, and possible risk factors for, the various complications of leprosy, including relapse, reactions and nerve function impairment. The methods of diagnosis, classification and treatment with MDT are described; nerve function was assessed at every visit to the clinic using a standardized methodology, so that reactions and new impairment could be detected early and treated. Eighty-four per cent of new case had at least one thickened nerve, with the ulnar nerve most commonly involved. Seventy-seven per cent of cases completed treatment and only one adverse reaction to the MDT drugs was noted. Twenty-eight per cent of all patients were given steroids at one time or another, almost always for new nerve function impairment, and 3% of these developed significant complications of steroid treatment. Twenty-nine patients (5%) received hospital care, including 14 patients who underwent major surgery. Sixty-one per cent of the women over 19 years of age had at least one pregnancy, but pregnancies were much less common after leprosy was diagnosed.

The pattern of leprosy-related neuropathy in the AMFES patients in Ethiopia: definitions, incidence, risk factors and outcome.

The ALERT MDT Field Evaluation Study (AMFES) began in 1988 and followed patients prospectively for up to 10 years after release from treatment (RFT). This paper presents the findings from this cohort with regard to neuropathy and nerve damage. Five hundred and ninety-four new cases of leprosy are included in the study, 300 multibacillary (MB) and 294 paucibacillary (PB) cases. Fifty-five percent of patients had some degree of impairment at diagnosis and a further 73 (12%) developed new nerve function impairment (NFI) after starting multiple drug therapy (MDT). The overall incidence rate for neuropathy was 39 episodes per 100 PYAR in the first year after diagnosis, gradually declining to 12 episodes per 100 PYAR in the sixth year. In those patients without impairment at diagnosis, the incidence rate of neuropathy was 25 episodes per 100 PYAR for MB cases and 11 per 100 PYAR for PB cases in the first year; in 33% of MB cases whose first episode of neuropathy occurred after diagnosis, that first episode took place after the first year, or after the normal period of treatment with MDT. Seventy-three patients with neuropathy developing after diagnosis are reported more fully: 34 (47%) had only one nerve involved and of these 25 (73%) had a single, acute episode of neuropathy. Nine (27%) had further episodes. Thirty-nine (53%) had more than one nerve involved and of these 16 (41%) had a single, acute episode, while 23 (59%) had further episodes. The terms 'chronic' and 'recurrent' neuropathy are defined and used to describe the pattern of neuropathy in those with repeated attacks. In patients with no impairment at the start of the study, treatment with steroids resulted in full recovery in 88% of nerves with acute neuropathy but only 51% of those with chronic or recurrent neuropathy. The median time to full recovery from acute neuropathy was approximately 6 months, but in a few cases recovery occurred gradually over 2-3 years. Severe neuropathy was less likely to be followed by a complete recovery than mild or moderate neuropathy. Forty-two percent of nerves with acute neuropathy that were not treated with steroids also fully recovered. In the group of patients who were thought to have old, permanent impairments at diagnosis, full recovery of nerve function occurred in 87/374 (23%) of the nerves involved. The overall outcome is illustrated by examining the average EHF score for groups of patients. Patients with no new neuropathy after diagnosis show a gradual improvement in their EHF score, while those with any episodes of neuropathy after diagnosis show a gradual deterioration after completion of MDT. Possible explanations for these findings are discussed. Risk factors for neuropathy, for chronic and recurrent neuropathy, and for a poor outcome 5 years after release from treatment, are examined. Impairment at diagnosis was the main risk factor for a poor outcome, accompanied by the occurrence of chronic/recurrent neuropathy or a reversal reaction.

Reversal reactions in the skin lesions of AMFES patients: incidence and risk factors.

Reversal reactions affect the skin and/or nerves of leprosy patients. This paper looks at reversal reactions involving the skin in 594 new patients in central Ethiopia, followed for between 6 and 11 years after the start of treatment. The incidence of reversal reaction declines steadily after the start of treatment, but the first episode may occur as long as 5 years after diagnosis in both paucibacillary (PB) and multibacillary (MB) patients. Recurrent episodes occurred up to 6 years after diagnosis. PB patients were at greatest risk for reversal reaction in the first year after diagnosis and MB patients in the first 4 years. The highest incidence rate was 18 episodes per 100 person years in MB patients during the first year after diagnosis. The ratio of the incidence rates for the first 3 years in MB versus PB patients is 2.4 (95% CI 1.6-3.8). This study confirms that starting effective treatment and borderline classification are risk factors for reversal reactions. Pregnancy/delivery in the 6 months prior to diagnosis was a significant risk factor for presenting with a reversal reaction [relative risk (RR) 5.9 (95% CI 2.1-16.5)], but later pregnancies were not associated with an increased risk. Being female was a significant risk factor for the late appearance of the first episode of reversal reaction. Having a reversal reaction in the first year after diagnosis was a highly significant risk factor for the development of later reactions [RR in PB cases 11.9 (95% CI 3.4-41.7); in MB cases 6.4 (95% CI 3.8-10.6)]. Being HIV positive was a risk factor for developing recurrent reversal reactions, although only three out of 29 recurrent cases were HIV positive [RR 2.7 (95% CI 1.4-5.1)].

ENL reactions in the multibacillary cases of the AMFES cohort in central Ethiopia: incidence and risk factors.

Erythema nodosum leprosum (ENL), or type 2 leprosy reactions are an important complication of multibacillary leprosy. The AMFES cohort includes 300 new multibacillary cases that have been followed for up to 10 years from the start of treatment, in central Ethiopia. Sixteen (5.3%) patients had ENL reactions. The incidence of ENL was maximal in the second and third years after the start of treatment, reaching 6.9 episodes per 100 person years at risk. Factors associated with being lepromatous [LL classification and a high bacillary index (BI)] gave an increased risk of developing ENL; in the univariate analysis, LL classification gave a relative risk of 3.6 (95% CI 1.3-10) and a BI of 6 gave a relative risk of 8.6 (95% CI 2.3-32) for the development of ENL. HIV co-infection was found to be a risk factor in this cohort, but as the numbers involved are small (only two HIV positive patients had ENL), this finding must be confirmed in larger studies. Ten of the 16 cases had recurrent episodes and five had at least five episodes occurring over a period of more than 2 years. The management and prognosis of ENL reactions are discussed.

Relapses after fixed duration multiple drug therapy: the AMFES cohort.

Relapse rates after multiple-drug therapy (MDT) have been low, although there remains a concern about the possibility of late relapse in those with an initially high bacterial load. In all, 502 patients in the AMFES cohort completed fixed-duration MDT and are included in this report. There have been no confirmed relapses in the AMFES cohort, in a follow-up period of up to 8 years after completion of treatment, even in the 57 cases with an initial average bacillary index of > or = 4.0, 20 of whom have been followed for more than 5 years after ceasing MDT. Methods of diagnosing a relapse are discussed.

The effect of HIV status on the clinical picture of leprosy: a prospective study in Ethiopia.

No major interaction between HIV infection and leprosy has been documented. The ALERT MDT Field Evaluation Study (AMFES) has allowed the examination of possible interactions in a prospective manner, although the total number of HIV-positive individuals was not high at 22 (3.8%) of 581 patients tested. There was an excess number of deaths in the HIV-positive group: 27% compared with 5.7% in the HIV-negative group, although the causes of death were not recorded (relative risk 4.8; 95% CI 2.2-10.2). HIV-positive individuals had a higher risk of ENL reactions (relative risk 5.2; 95% CI 1.7-15.9). Reversal reactions and neuritis (both acute and chronic) were not significantly influenced by HIV status, although there was a possible increase in recurrent reversal reactions in HIV-positive cases (relative risk 2.2; 95% CI 0.98-4.7). There was no evidence to suggest an increased risk of developing leprosy or of developing multibacillary rather than paucibacillary disease. There was no association between HIV positivity and the development of impairment.

The pattern of decline in bacillary index after 2 years of WHO recommended multiple drug therapy: the AMFES cohort.

With effective antibiotic treatment, the bacillary index (BI) in multibacillary leprosy patients declines over a number of years. This can be quantified as a rate of decline in log-units per year or as the time until smear negativity is reached. In the AMFES cohort 220 cases had data on the changes in their BI over time, while 170 cases are documented until smear negativity. The average BI at the start was 3.3 (SD 1.5; range 0.3-5.5) and the mean rate of decline was 0.85 units per year (median 0.7 units per year); in the first 2 years after diagnosis, the mean rate of decline was 1.15 units per year. The rate of decline was not related to any clinical features of the disease except delay in diagnosis: patients presenting for treatment early had a significantly faster rate of clearing the bacilli (adjusted relative risk 2.3; 95% CI 1.0-5.1). Fifty-eight percent of cases took longer than 3 years to reach smear negativity, but this time interval is largely determined by the initial BI and classification, making it a less useful indicator of bacterial clearance. More severe impairment at the start of treatment was associated with a faster return to smear negativity, for which no obvious explanation can be given. Reversal reactions, which occurred in 25% of the cases reviewed, are not associated with a more rapid clearance of bacilli.

The hand-foot impairment score as a tool for evaluating prevention of disability activities in leprosy: an exploration in patients treated with corticosteroids.

The hand-foot (HF) impairment score in leprosy patients is the sum of the WHO disability grades for hands and feet. This retrospective study explored the possibility of using the HF score for evaluation of the effectiveness of corticosteroid treatment programmes for nerve function impairment (NFI). Changes in the score were compared with changes in sensory testing (ST) and voluntary muscle testing (VMT) for 42 leprosy patients who received corticosteroid treatment. The WHO grade did not change in 30/60 (50%) of extremities gaining, and in 4/10 (40%) extremities losing sensation and/or muscle strength. However, 18/24 (75%) patients with a definite gain in function improved in HF score, while the HF score remained unchanged in 10/11 (91%) patients with no change in nerve function. Five patients with impairment in multiple extremities showed both gain and loss of sensation and/or muscle strength in the same or different extremities. Overall, improvement, deterioration and absence of change in NFI, as indicated by changes in ST and VMT were reflected correctly by the HF score in 28 (76%) of the remaining 37 patients. It was also shown that the HF score does not give appropriate information on the extent of the effect of corticosteroid treatment. This study illustrates that the HF score can not be used to support management of corticosteroid treatment of individual patients, but indicates this score to be a promising device for the evaluation of the effectiveness of corticosteroid treatment programmes. This study used the HF score because information on (changes in) eye impairment was not considered reliable. However, in principle, we consider the EHF score, which is the sum of the WHO disability grades for hands, feet and eyes, preferable for evaluation purposes. We strongly recommend further validation of the EHF score as a tool for evaluation of corticosteroid treatment programmes for patient groups with different distributions of NFI through prospective studies.

Which physical signs help most in the diagnosis of leprosy? A proposal based on experience in the AMFES project, ALERT, Ethiopia.

As integration of leprosy control programmes proceeds, general health staff will have responsibility for the diagnosis of most new cases of leprosy. The training required by these workers has not yet been set out in detail. In this paper the criteria for making the diagnosis of leprosy in the AMFES cohort of 594 new cases are examined. Since this study does not include details of suspects in whom leprosy was excluded on clinical grounds, true sensitivity and specificity values cannot be calculated, but the positive predictive value of the diagnostic criteria can be measured. Sensory loss in a typical skin patch is the most important sign of early leprosy, but was not present in 132 (49%) of the 268 cases with a positive skin smear. Thickening of the ulnar nerve is a valuable sign of leprosy in Ethiopia. It can be taught to health workers, who can practise by examining their own ulnar nerves. It is more likely to be present than nerve function impairment and is particularly important when skin smears are difficult to do or are unreliable. We recommend that five basic signs are used, the presence of any two being diagnostic of leprosy: Skin lesion(s) consistent with leprosy. Loss of sensation in such a lesion. Thickening of either ulnar nerve. Loss of sensation in the palm of the hand or the sole of the foot. The presence of acid-fast bacilli in skin smears. Exact policies for the diagnosis of leprosy should be worked out and validated for each national programme.