Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis.

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.

Comparative evaluation of antibody detection tests to facilitate the diagnosis of multibacillary leprosy.

Despite control efforts, leprosy persists as a significant health concern in many regions. Diagnosis is achieved by a combination of clinical, histopathological, and bacteriological examinations, each of which presents a barrier to expeditious diagnosis, particularly by non-experts. Immunological investigations in research laboratories have clearly indicated that antibody detection tests could aid the diagnosis of leprosy. In this study, we detected circulating antibodies with two rapid diagnostic tests (RDT) involving immunochromatographic lateral flow platforms and one rapid ELISA system. Leprosy patients were identified with a high degree of sensitivity in each assay (over 80% in all; over 90% among cases with bacterial indices >1+), although critical differences were observed in specificity. While the specificity of CTK OnSite Leprosy Ab Rapid Test and InBios Leprosy Detect™ fast ELISA were high (96.4 and 93.7% in the general population, respectively), there was a marked reduction in OrangeLife NDO-LID® RDT (only 25.0%). As anticipated, seropositivity rates were marginally higher in contacts of leprosy patients than in endemic controls. Although we observed a slight drop in test band intensity when blood, rather than serum, was used to develop OnSite Leprosy Ab Rapid Tests, the sensitivity and specificity of these tests was unaffected. When we contrasted test performance with clinical and bacteriological information, we found that RDT and ELISA results positively correlated with the bacteriological index. These data indicate that these assays could be a ready replacement of invasive, insensitive, and time consuming skin slit smear procedures that additionally require expert microscopic examinations. We propose that, due to their speed and point of care applicability, the RDT could be used as an initial entry point to the diagnostic protocols, with confirmation of results attained in a highly quantitative manner following serum transfer to a reference laboratory.

Need for, and acceptability of, rapid diagnostic tests that can facilitate the diagnosis of leprosy.

New cases of leprosy indicate that M. leprae transmission is continuing. In the first half of 2015 we sequentially diagnosed new leprosy patients attending Cebu Skin Clinic, a referral and leprosy treatment centre in Cebu City, Philippines. The great majority of patients were characterised as multibacillary (145 of 147; 98·6%), most of whom had bacterial indices 3þ (86; 58·5%) or more than 20 skin lesions (94; 63·9%). Importantly, 65·3% estimated that slightly over a year had elapsed between their initial symptoms and diagnosis, while 26·5% reported a lapse of greater than 3 years. Many patients (73·8%) and their household contacts (79·2%) reported that their lives had already been adversely affected. This indicates that improvements to expedite diagnosis are required, and rapid diagnostic tests (RDT) appear suited for this. Questionnaires were conducted among various groups to ascertain the acceptability and perception of RDT. All groups responded favourably, with 95·9% patients, 93·2% contacts and 81·4% of the general population responding that RDT would be beneficial for leprosy. The vast majority of patients (88·6%) indicated that they would 'definitely' submit to testing, followed by 69·4% of contacts and 72·2% of the general population. The majority of patients thought their household members should also be tested, while a subset indicated that RDT use should be extended to their respective communities. We propose that, due to their ease of use, point of care applicability and acceptability within target populations, RDT could be used as an entry point to inform the diagnostic process for leprosy.

Rapid quantitative serological test for detection of infection with Mycobacterium leprae, the causative agent of leprosy.

Leprosy remains an important health problem in a number of regions. Early detection of infection, followed by effective treatment, is critical to reduce disease progression. New sensitive and specific tools for early detection of infection will be a critical component of an effective leprosy elimination campaign. Diagnosis is made by recognizing clinical signs and symptoms, but few clinicians are able to confidently identify these. Simple tests to facilitate referral to leprosy experts are not widely available, and the correct diagnosis of leprosy is often delayed. In this report, we evaluate the performance of a new leprosy serological test (NDO-LID). As expected, the test readily detected clinically confirmed samples from patients with multibacillary (MB) leprosy, and the rate of positive results declined with bacterial burden. NDO-LID detected larger proportions of MB and paucibacillary (PB) leprosy than the alternative, the Standard Diagnostics leprosy test (87.0% versus 81.7% and 32.3% versus 6.5%, respectively), while also demonstrating improved specificity (97.4% versus 90.4%). Coupled with a new cell phone-based test reader platform (Smart Reader), the NDO-LID test provided consistent, objective test interpretation that could facilitate wider use in nonspecialized settings. In addition, results obtained from sera at the time of diagnosis, versus at the end of treatment, indicated that the quantifiable nature of this system can also be used to monitor treatment efficacy. Taken together, these data indicate that the NDO-LID/Smart Reader system can assist in the diagnosis and monitoring of MB leprosy and can detect a significant number of earlier-stage infections.

Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy.

Although leprosy is curable with drug treatment, the identification of biomarkers of infection, disease progression and treatment efficacy would greatly help to reduce the overall prevalence of the disease. Reliable biomarkers would also reduce the incidence of grade-2 disability by ensuring that those who are most at risk are diagnosed and treated early or offered repeated treatments in the case of relapse. In this study, we examined the reactivity of sera from lepromatous and tuberculoid leprosy patients (LPs) against a panel of 12 recombinant Mycobacterium leprae proteins and found that six proteins were strongly recognised by multibacillary (MB) patients, while only three were consistently recognised by paucibacillary patients. To better understand the dynamics of patient antibody responses during and after drug therapy, we measured antibody titres to four recombinant proteins, phenolic glycolipid-I and lipoarabinomannan at baseline and up to two years after diagnosis to investigate the temporal changes in the antibody titres. Reactivity patterns to individual antigens and decreases in antibody titres were patient-specific. Antibody titres to proteins declined more rapidly vs. those to carbohydrate and glycolipid antigens. Compared to baseline values, increases in antibody titres were observed during reactional episodes in one individual. Additionally, antibody responses against a subset of antigens that provided a good prognostic indicator of disease progression were analysed in 51 household contacts of MB index cases for up to two years. Although the majority of these contacts showed no change or exhibited decreases in antibody titres, seven individuals developed higher titres towards one or more of these antigens and one individual with progressively higher titres was diagnosed with borderline lepromatous leprosy 19 months after enrolment. The results of this study indicate that antibody titres to specific M. leprae antigens can be used to monitor treatment efficacy in LPs and assess disease progression in those most at risk for developing this disease.

A Sensitive and Quantitative Assay to Enumerate and Measure Mycobacterium leprae Viability in Clinical and Experimental Specimens.

Mycobacterium leprae, the etiologic agent of leprosy, cannot be cultured on artificial media. This characteristic, coupled with its long generation time, presents a number of unique challenges to studying this pathogen. One of the difficulties facing both researchers and clinicians is the absence of a rapid test to measure the viability of M. leprae in clinical or experimental specimens. The lack of such a tool limits the understanding of M. leprae immunopathogenesis and makes determining the efficacy of drug treatments difficult. With this in mind, we developed a robust two-step molecular viability assay (MVA) that first enumerates the M. leprae in the tissue; then, this data is used to normalize bacterial RNA quantities for the second step, in which the expression of M. leprae esxA and hsp18 are measured. This assay is specific and sensitive enough to be used on most clinical samples. This protocol describes the steps required to extract DNA and RNA from M. leprae-infected tissue, enumerate M. leprae, and measure M. leprae viability based on the normalized expression of two M. leprae-specific genes (hsp18 and esxA). This protocol also outlines an optimal laboratory design and workflow for performing this assay. © 2022 The Leprosy Mission Nepal. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: DNA and RNA P purification from M. leprae-infected tissue Basic Protocol 2: Enumeration of M. leprae by RLEP qPCR on the DNA fraction Basic Protocol 3: Calculation of M. leprae per tissue and normalization of RNA Basic Protocol 4: Reverse-transcription of normalized RNA to generate cDNA Basic Protocol 5: Determination of M. leprae viability using HSP18 and ESXA qPCR on the cDNA Support Protocol 1: M. leprae qPCR primer/probe stock preparation Support Protocol 2: Preparation of plasmid stocks and standard curves.

The efficacy of a four-week, ofloxacin-containing regimen compared with standard WHO-MDT in PB leprosy.

OBJECTIVES: To compare the efficacy of a 4-week ofloxacin-containing regimen and the standard WHO-MDT regimen for PB leprosy, in terms of the rate and timing of relapse after treatment completion. DESIGN: 124 PB patients were enrolled in a randomised, double-blind trial. Of these, 66 received the standard 6-month WHO-MDT regimen, whereas 58 received 28 daily supervised doses of rifampicin 600mg + ofloxacin 400 mg, plus 5 months of placebo. Patients were regularly monitored for clinical response and for signs of relapse after treatment completion. RESULTS: Patients enrolled in the ofloxacin group had a mean follow-up of 10.8 years (628 patient-years) with 1 early relapse at 3 years after treatment completion. On relapse, this patient remained smear negative but was reclassified by current WHO criteria (> or =6 skin lesions) as multibacillary (MB). Patients on the WHO-MDT regimen had a mean follow-up of 11.3 years (749 patient-years) with two late relapses at 8 and 12 years, both still classified as PB on relapse. CONCLUSION: In conclusion, both regimens appeared generally efficacious, and, in particular, resulted in few relapses.

Utility and limitations of serodiagnostic tests in monitoring the response to treatment of leprosy patients.

Simple measures that can facilitate early recognition of leprosy complications are still lacking. We therefore evaluated a lateral flow-based rapid diagnostic test and fast enzyme-linked immunosorbent assay measuring anti-LID-NDO antibody responses among leprosy cases in Cebu, Philippines. Responses were measured at diagnosis, then during and after the provision of standard multidrug therapy. Our data indicate that both platforms are highly sensitive tools for the primary diagnosis of, in particular, multibacillary leprosy. A gradual, quantifiable decline in both magnitude of response and percent positive responders was observed during and after treatment. As a group, patients that developed erythema nodosum leprosum (ENL) had a significantly higher response at diagnosis than patients that either developed reversal reactions or did not develop reactions. Although higher initial anti-NDO-LID responses were a risk factor for ENL, neither platform, however, could reliably predict the time of emergence of reactional episodes.

Combination chemoprophylaxis and immunoprophylaxis in reducing the incidence of leprosy.

Leprosy is a complex infectious disease caused by Mycobacterium leprae that is a leading cause of nontraumatic peripheral neuropathy. Current control strategies, with a goal of early diagnosis and treatment in the form of multidrug therapy, have maintained new case reports at ~225,000 per year. Diagnostic capabilities are limited and even with revisions to multidrug therapy regimen, treatment can still require up to a year of daily drug intake. Although alternate chemotherapies or adjunct immune therapies that could provide shorter or simpler treatment regimen appear possible, only a limited number of trials have been conducted. More proactive strategies appear necessary in the drive to elimination. As a prevention strategy, most chemoprophylaxis campaigns to date have provided about a 2-year protective window. Vaccination, in the form of a single bacillus Calmette-Guérin (BCG) immunization, generally provides ~50% reduction in leprosy cases. Adapting control strategies to provide both chemoprophylaxis and immunoprophylaxis has distinct appeal, with chemoprophylaxis theoretically buttressed by vaccination to generate immediate protection that can be sustained in the long term. We also discuss simple assays measuring biomarkers as surrogates for disease development or replacements for invasive, but not particularly sensitive, direct measures of M. leprae infection. Such assays could facilitate the clinical trials required to develop these new chemoprophylaxis, immunoprophylaxis strategies, and transition into wider use.

A rapid ELISA for the diagnosis of MB leprosy based on complementary detection of antibodies against a novel protein-glycolipid conjugate.

Despite the widespread use of multidrug therapy for treatment, delays in clinical recognition and under-reporting of leprosy indicate that Mycobacterium leprae transmission is continuing. Thus, leprosy is likely to persist as a significant burden on health systems in many regions. In this study, we combined 2 previously characterized leprosy antigens, leprosy IDRI diagnostic-1 (LID-1) and ND-O, into the single fusion complex (ND-O-LID) and determined the serum antibody responses of leprosy patients from Colombia and the Philippines. Following confirmation that antibodies recognized each component within the conjugate, we assessed the performance of a rapid enzyme-linked immunosorbent assay (ELISA) system (Leprosy Detect(TM) fast ELISA; InBios International, Inc., Seattle, WA, USA) based on ND-O-LID capable of generating results within 1.5hours of sample addition. We found ELISA results correlated with the bacteriological index and Ridley-Jopling categorization, with lepromatous leprosy patients having the highest responses, while those of borderline tuberculoid patients were lower. Multibacillary (MB) leprosy patients were distinguished with a high degree of sensitivity (95.7%) and specificity (93.2%), suggesting that this ELISA could potentially replace invasive and insensitive skin slit smear procedures that require expert microscopic examinations. Due to the speed and robustness of this assay, we believe this is an excellent tool for detecting MB leprosy patients in a simple and highly-quantitative manner.

Long-term relapse risk of multibacillary leprosy after completion of 2 years of multiple drug therapy (WHO-MDT) in Cebu, Philippines.

From 1987 to 1994, we enrolled 500 subjects completing 2-year WHO multiple drug therapy (MDT) for multibacillary leprosy in a prospective relapse study. Relapse was defined as new skin lesions and an increase in the bacterial index (BI) > or = 2+ (> or = 100x) at any single slit-skin smear site. At the study end in 2006, follow-up was 6,401 subject-years, a mean of 12.8 years/subject. We observed 23 relapses, 6-16 years after MDT (mean, 10.5 years; 95% confidence interval [CI], 9.2-11.8), peaking in Years 11-12 (> 1%/year). The cumulative risk was 6.6% (95% CI, 5.0-8.2%). In a subset of 181 subjects with pre-MDT average BI > or = 4+, 11 relapses occurred (cumulative risk, 10.1%). In mouse footpad assays, Mycobacterium leprae from relapsed subjects were rifampin and clofazimine sensitive. Taken together, the data suggest relapses are related to activation of dormant organisms (persisters) not killed by MDT rather than new infection.

Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy

Although leprosy is curable with drug treatment, the identification of biomarkers of infection, disease progression and treatment efficacy would greatly help to reduce the overall prevalence of the disease. Reliable biomarkers would also reduce the incidence of grade-2 disability by ensuring that those who are most at risk are diagnosed and treated early or offered repeated treatments in the case of relapse. In this study, we examined the reactivity of sera from lepromatous and tuberculoid leprosy patients (LPs) against a panel of 12 recombinant Mycobacterium leprae proteins and found that six proteins were strongly recognised by multibacillary (MB) patients, while only three were consistently recognised by paucibacillary patients. To better understand the dynamics of patient antibody responses during and after drug therapy, we measured antibody titres to four recombinant proteins, phenolic glycolipid-I and lipoarabinomannan at baseline and up to two years after diagnosis to investigate the temporal changes in the antibody titres. Reactivity patterns to individual antigens and decreases in antibody titres were patient-specific. Antibody titres to proteins declined more rapidly vs. those to carbohydrate and glycolipid antigens. Compared to baseline values, increases in antibody titres were observed during reactional episodes in one individual. Additionally, antibody responses against a subset of antigens that provided a good prognostic indicator of disease progression were analysed in 51 household contacts of MB index cases for up to two years. Although the majority of these contacts showed no change or exhibited decreases in antibody titres, seven individuals developed higher titres towards one or more of these antigens and one individual with progressively higher titres was diagnosed with borderline lepromatous leprosy 19 months after enrolment. The results of this study indicate that antibody titres to specific M. leprae antigens can be used to monitor treatment efficacy in LPs and assess disease progression in those most at risk for developing this disease.