Zoonotic Leprosy in the Southeastern United States

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and have been implicated in zoonotic transmission of leprosy. Early studies found this disease mainly in Texas and Louisiana, but armadillos in the southeastern United States appeared to be free of infection. We screened 645 armadillos from 8 locations in the southeastern United States not known to harbor enzootic leprosy for M. leprae DNA and antibodies. We found M. leprae–infected armadillos at each location, and 106 (16.4%) animals had serologic/PCR evidence of infection. Using single-nucleotide polymorphism variable number tandem repeat genotyping/genome sequencing, we detected M. leprae genotype 3I-2-v1 among 35 armadillos. Seven armadillos harbored a newly identified genotype (3I-2-v15). In comparison, 52 human patients from the same region were infected with 31 M. leprae types. However, 42.3% (22/52) of patients were infected with 1 of the 2 M. leprae genotype strains associated with armadillos. The geographic range and complexity of zoonotic leprosy is expanding.     

Dynamics of Mycobacterium leprae transmission in environmental context: Deciphering the role of environment as a potential reservoir

Leprosy is a disease caused by Mycobacterium leprae. Various modes of transmission have been suggested for this disease. Transmission and risk of the infection is perhaps related to presence of the infectious cases and is controlled by environmental factors. Evidence suggests that humidity may favor survival of M. leprae in the environment. Several reports show that non-human sources like ‘naturally’ infected armadillos or monkeys could act as reservoir for M. leprae. Inanimate objects or fomites like articles used by infectious patients may theoretically spread infection. However, it is only through detailed knowledge of the biodiversity and ecology that the importance of this mode of transmission can be fully assessed. Our study focuses here to decipher the role of environment in the transmission of the disease. Two hundred and seven soil samples were collected from a village in endemic area where active cases also resided at the time of sample collection. Slit skin smears were collected from 13 multibacillary (MB) leprosy patients and 12 household contacts of the patients suspected to be hidden cases. DNA and RNA of M. leprae were extracted and amplified using M. leprae specific primers. Seventy-one soil samples showed presence of M. leprae DNA whereas 16S rRNA could be detected in twenty-eight of these samples. Samples, both from the environment and the patients, exhibited the same genotype when tested by single nucleotide polymorphism (SNP) typing. Genotype of M. leprae found in the soil and the patients residing in the same area could help in understanding the transmission link in leprosy.     

BCG-induced immunity profiles in household contacts of leprosy patients differentiate between protection and disease

Leprosy is an infectious disease caused by Mycobacterium leprae leading to irreversible disabilities along with social exclusion. Leprosy is a spectral disease for which the clinical outcome after M. leprae infection is determined by host factors. The spectrum spans from anti-inflammatory T helper-2 (Th2) immunity concomitant with large numbers of bacteria as well as antibodies against M. leprae antigens in multibacillary (MB) leprosy, to paucibacillary (PB) leprosy characterised by strong pro-inflammatory, Th1 as well as Th17 immunity. Despite decades of availability of adequate antibiotic treatment, transmission of M. leprae is unabated. Since individuals with close and frequent contact with untreated leprosy patients are particularly at risk to develop the disease themselves, prophylactic strategies currently focus on household contacts of newly diagnosed patients.It has been shown that BCG (re)vaccination can reduce the risk of leprosy. However, BCG immunoprophylaxis in contacts of leprosy patients has also been reported to induce PB leprosy, indicating that BCG (re)vaccination may tip the balance between protective immunity and overactivation immunity causing skin/nerve tissue damage.In order to identify who is at risk of developing PB leprosy after BCG vaccination, amongst individuals who are chronically exposed to M. leprae, we analyzed innate and adaptive immune markers in whole blood of household contacts of newly diagnosed leprosy patients in Bangladesh, some of which received BCG vaccination. As controls, individuals from the same area without known contact with leprosy patients were similarly assessed.Our data show the added effect of BCG vaccination on immune markers on top of the effect already induced by M. leprae exposure. Moreover, we identified BCG-induced markers that differentiate between protective and disease prone immunity in those contacts.     

A migration-driven model for the historical spread of leprosy in medieval Eastern and Central Europe

Leprosy was rare in Europe during the Roman period, yet its prevalence increased dramatically in medieval times. We examined human remains, with paleopathological lesions indicative of leprosy, dated to the 6th–11th century AD, from Central and Eastern Europe and Byzantine Anatolia. Analysis of ancient DNA and bacterial cell wall lipid biomarkers revealed Mycobacterium leprae in skeletal remains from 6th–8th century Northern Italy, 7th–11th century Hungary, 8th–9th century Austria, the Slavic Greater Moravian Empire of the 9th–10th century and 8th–10th century Byzantine samples from Northern Anatolia. These data were analyzed alongside findings published by others. M. leprae is an obligate human pathogen that has undergone an evolutionary bottleneck followed by clonal expansion. Therefore M. leprae genotypes and sub-genotypes give information about the human populations they have infected and their migration. Although data are limited, genotyping demonstrates that historical M. leprae from Byzantine Anatolia, Eastern and Central Europe resembles modern strains in Asia Minor rather than the recently characterized historical strains from North West Europe. The westward migration of peoples from Central Asia in the first millennium may have introduced different M. leprae strains into medieval Europe and certainly would have facilitated the spread of any existing leprosy. The subsequent decline of M. leprae in Europe may be due to increased host resistance. However, molecular evidence of historical leprosy and tuberculosis co-infections suggests that death from tuberculosis in leprosy patients was also a factor.     

Experimental chemotherapy in leprosy

The Memorandum reviews the considerable progress that has been made in research on the chemotherapy of leprosy during the last 10-15 years, as a result of which it is now possible to study the same topics in leprosy as are studied in other bacterial diseases. Thus drugs have been screened in mice for their activity against Mycobacterium leprae. Those that have been found to have the greatest activity against M. leprae at acceptable dosages—dapsone, rifampicin, and clofazimine—have been characterized in terms of the minimal effective dosage and rate of bacterial kill. Similarly, their pharmacokinetics in man and in certain animals have been defined. The theoretical basis for drug trials in leprosy patients is discussed in terms of the number of viable and the number of dead M. leprae that remain at various stages of therapy.     

Towards the molecular epidemiology of Mycobacterium leprae: Strategies, successes, and shortcomings

Mycobacterium leprae, the causative agent of leprosy, is an unusual organism that presents unique challenges to those studying the disease through molecular epidemiology. As a consequence, many basic aspects of disease transmission and biology remain unilluminated. In this review, we explore the general principles of molecular epidemiology, and the special difficulties surrounding the application of molecular epidemiology to M. leprae. We briefly discuss the computational tools commonly employed in molecular epidemiology studies. The past decade of developments in molecular strain typing approaches through VNTRs and SNP loci, and their merits and limitations, are discussed. We summarize what has been learned about the transmission and historical origins of leprosy through molecular epidemiology and Bacterial Population Genetics, to date. Lastly, we critically evaluate the strengths and shortcomings of leprosy research, and present recommendations for future work that will hopefully shed light on some of the disease’s most fundamental mysteries.     

Development and pre-clinical assessment of a 73 kD chimeric fusion protein as a defined sub-unit vaccine for leprosy

Despite the advances toward the elimination of leprosy through widespread provision of multi-drug therapy to registered patients over the last 2 decades, new case detection rates have stabilized and leprosy remains endemic in a number of localized regions. A vaccine could overcome the inherent limitations of the drug treatment program by providing protection in individuals who are not already harboring the Mycobacterium leprae bacilli at the time of administration and effectively interrupt the transmission cycle over a wider timespan. In this report we present data validating the production of 73f, a chimeric fusion protein incorporating the M. leprae antigens ML2028, ML2346 and ML2044. The 73f protein was recognized by IgG in multibacillary (MB) leprosy patient sera and stimulated IFNγ production within whole blood assays of paucibacillary (PB) leprosy patient and healthy household contacts of MB patients (HHC). When formulated with a TLR4L-containing adjuvant (GLA-SE), 73f stimulated a strong and pluripotent Th1 response that inhibited M. leprae-induced inflammation in mice. We are using these data to develop new vaccine initiatives for the continued and long-term control of leprosy.     

Cutaneous leishmaniasis and leprosy

Eight patients who had concomitant leprosy and leishmaniasis are described. Two patients with lepromatous leprosy had high resistance leishmaniasis, implying that the immune deficiency in lepromatous leprosy is specific to Mycobacterium leprae.     

NLRP1 haplotypes associated with leprosy in Brazilian patients

Polymorphisms in innate immunity genes are known to be involved in the multifactorial susceptibility to Mycobacterium leprae infection. M. leprae can downregulate IL-1β secretion escaping monocyte digestion. The intracellular receptors NLRPs (NACHT, LRR and PYD domains-containing proteins) sense pathogen associated molecular patterns (PAMPs) activating caspase-1 and IL-1β secretion in the context of inflammasome. Considering the possible role of inflammasome in the immune response against M. leprae, known single nucleotide polymorphisms (SNPs) in two NLRP genes, NLRP1 and NLRP3, were analyzed in Brazilian leprosy patients.Disease-associated SNPs (5 in NLRP1 and 2 in NLRP3), previously associated to infections and to immunologic disorders, were genotyped in 467 leprosy patients (327 multibacillary, MB; 96 paucibacillary, PB) and in 380 healthy controls (HC) from the state of Sao Paulo (Brazil), and in 183 patients (147 MB; 64 PB) and 186 HC from Mato Grosso (Brazil). Logistic regression analysis was performed considering susceptibility to leprosy di per se (leprosy versus HC) and clinical form (MB versus PB), adjusting for gender and ethnicity.Whereas none of the considered SNPs were statistically associated with leprosy, the NLRP1 combined haplotype rs2137722/G-rs12150220/T-rs2670660/G resulted significantly more frequent in patients than in HC as well as in PB than in MB. While both associations were lost after correction for gender and ethnicity, the NLRP1 combined haplotype rs2137722/G-rs12150220/A-rs2670660/G resulted strongly associated to PB.NLRP1 might be involved in the susceptibility to leprosy with particular emphasis for PB clinical form. Although preliminary, this is the first report linking NLRPs and inflammasome with leprosy: replication studies as well as functional assays are envisaged to deeper investigate the role of NLRP1 in M. leprae infection. Interestingly, NLRP1 SNPs were previously associated to susceptibility to Crohn disease, suggesting that NLRP1 could be a new modifier gene in common between leprosy and Crohn disease.     

Genomic diversity in Mycobacterium leprae isolates from leprosy cases in South India

ObjectiveThe Objective of this study was to identify the strain diversity of Mycobacterium leprae in terms of SNP types and subtypes stratified as per genomic single nucleotide polymorphisms, in clinical isolates of leprosy patients from a tertiary care leprosy center in South India. Further, the associations of SNP types with clinical outcomes in leprosy were also investigated.MethodsDNA was extracted from excisional skin biopsies of a total of 172 newly diagnosed untreated leprosy patients from a clinic in Tamil Nadu, in south India, that also serves patients from neighboring states. All the leprosy patients were those who voluntarily reported at the clinic during the study period of one year i.e., 2015. Clinical and histopathological details were collected at diagnosis and leprosy was confirmed through bacteriological smear examination and PCR for M. leprae specific RLEP region. SNP types and subtypes were determined by PCR amplification and Sanger sequencing of PCR products.ResultsM. leprae specific RLEP gene amplification was achieved in 160 out of 172 patients. Among 160 specimens 118(73.75%) were type 1 and 42 (26.25%) were type 2 and on subtyping it was noted that 88/160 (55.00%) were 1D, 25/160 (15.62%) 1C, 5/160 (3.12%) 1A, 33/160 (20.62%) 2G and 9/160 (5.62%) were 2H.ConclusionOur results indicated that subtype 1D is predominant in the south Indian population. We also noted 2G, 1C and 1A in the patient sample tested. Additionally we identified subtype 2H for the first time in India.     

Seropositivity to anti-phenolic glycolipid-I in leprosy cases, contacts and no known contacts of leprosy in an endemic and a non-endemic area in northeast Brazil

The seroprevalence rates of IgM anti-phenolic glycolipid-I (PGL-I) antibodies in four study groups with differing exposure to Mycobacterium leprae in Ceará, Brazil were investigated between March 2005 and August 2006. The first three groups in a high prevalence area included 144 cases of leprosy, their 380 contacts and 317 participants with no known leprosy contact. The fourth group in a low prevalence area consisted of 87 participants with no known leprosy contact living in an area in which no cases of leprosy had been reported in the previous 6 months. Seropositivity and levels of IgM antibodies to PGL-I were investigated using ELISA. The seropositivity levels of anti-PGL-I among the different clinical forms of leprosy cases were 61% for lepromatous, 25% for tuberculoid and 27% indeterminate. The levels of anti-PGL-I antibodies in the endemic area differentiated leprosy cases from non-cases. However, the seropositivity was similar among contact cases (15.8%) and no known leprosy contact cases from high (15.1%) and low (13.8%) prevalence areas. The seropositivity of both contacts and no known contacts was much higher than previously reported among no known contacts in other endemic areas. The study indicates that anti-PGL-I antibodies are not useful as immunological markers of household leprosy contacts and no known leprosy contacts in endemic areas.     

Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India

Non-tuberculous mycobacteria (NTM) are environmental mycobacteria found ubiquitously in nature. The present study was conducted to find out the presence of various species of NTM in leprosy endemic region along with Mycobacterium (M) leprae. Water and wet soil samples from the periphery of ponds used by the community were collected from districts of Purulia of West Bengal and Champa of Chhattisgarh, India. Samples were processed and decontaminated followed by culturing on Lowenstein Jensen (LJ) media. Polymerase chain reaction (PCR) was performed using 16S rRNA gene target of mycobacteria and species was confirmed by sequencing method. Indirect immune-fluorescent staining of M. leprae from soil was performed using M. leprae-PGL-1 rabbit polyclonal antibody. The phylogenetic tree was constructed by using MEGA-X software. From 380 soil samples 86 NTM were isolated, out of which 34(40%) isolates were rapid growing mycobacteria (RGM) and 52(60%) isolates were slow growing mycobacteria (SGM). Seventy-seven NTM isolates were obtained from 250 water samples, out of which 35(45%) were RGM and 42(55%) were SGM. Amongst all the RGM, we isolated M. porcinum, M. psychrotolerans, M. alsenase, M. arabiense and M. asiaticum from Indian environmental samples. M. fortuitum was the most commonly isolated species of all RGM. Out of all SGM, M. holsaticum, M. yongonense, M. seoulense, M. szulgai, M. europaeum, M. simiae and M. chimaera were isolated for the first time from Indian environment. M. intracellulare was the commonest of all isolated SGM. Presence of M. leprae was confirmed by indirect immunofluorescent microcopy and PCR method from the same environmental samples. Phylogenetic tree was showing a close association between these NTMs and M. leprae in these samples. Several NTM species of pathogenic and nonpathogenic in nature along with M. leprae were isolated from soil and pond water samples from leprosy endemic regions and these might be playing a role in causing disease and maintaining leprosy endemicity in India.     

New prospects for the study of leprosy in the laboratory

Although Mycobacterium leprae was identified earlier than Myco. tuberculosis, it has still not been cultured in vitro and only in 1960 was an infection obtained in laboratory animals. However, important advances have been made in the field of experimental leprosy in the last decade due to the development of new techniques and models for studying Myco. leprae in vivo, thus overcoming the limitations imposed by a non-cultivable mycobacterium. Quantitative techniques using Myco. lepraemurium provided the first model for developing an indirect method for distinguishing dead (non-infectious) from living (infectious) bacilli, based on morphological differences in organisms stained by the Ziehl—Neelsen method. However, the most important advances resulted from the limited and localized growth of Myco. leprae when inoculated into the foot pads of mice and, later, the more substantial and generalized multiplication of Myco. leprae in immunologically deficient mice (thymectomized and irradiated with a dose of 900 r). Moreover, in the immunologically deficient animals, the infection eventually resulted in a disease replicating that of lepromatous type leprosy in man, including the involvement of peripheral nerves.     

A serological test for leprosy based on competitive inhibition of monoclonal antibody binding to the MY2a determinant of Mycobacterium leprae

A novel serological assay for leprosy has been devised on the basis of serum inhibition of binding of ¹²⁵I-labelled MLO4 monoclonal antibody to Mycobacterium leprae sonicate-coated microtitre plates. Antibodies were detected in 93% of lepromatous leprosy patients, whereas controls from the endemic area, including leprosy contacts and patients with tuberculosis, were serologically negative. The specificity and efficacy of the test may offer an advantage over previously used techniques.     

The problem of cultivation of Mycobacterium leprae. A review with criteria for evaluating recent experimental work

Some criteria are presented to help evaluate papers appearing in the literature claiming successful cultivation of M. leprae either in the absence or in the presence of tissue-cultured cells. Recently, electron microscopic studies have definitely shown M. leprae to belong to the genus Mycobacterium and its division to occur through transverse section. A survey is given of the mycobacterial strains isolated in the last 10 years from leprosy lesions. These strains belong to taxonomically different species and cannot be considered to be M. leprae. No substantiated claim was made concerning the in vitro growth of M. leprae and the application of the tissue culture technique has been equally disappointing. The view is expressed that progress towards the in vitro cultivation of M. leprae can be made only as a result of increased knowledge about the intracellular environment and the metabolic activities of this organism, to be obtained by the application of modern biochemical and histochemical techniques.     

Immunological determination of Mycobacterium leprae by means of cytoplasmic antigens

Mycobacterium leprae was isolated and purified from lepromas, the spleen, and the liver of leprosy patients. An immunodiffusion analysis of the cytoplasms obtained from four lots of M. leprae and M. lepraemurium, 295 strains of different actinomycetales, and 12 other bacteria was performed with the use of the cytoplasm antisera. Immunological relationships were revealed between the cytoplasms of M. leprae, M. lepraemurium, M. avium, M. gallinarum, M. tuberculosis, M. simiae, M. kansasii, M. chitae, M. capsulatum, Actinomyces israelii, A. naeslundii, and some strains of saprophytic mycobacteria. These studies led to the proposed concept of the immunological evolution of M. leprae and M. lepraemurium and an Actinomyces-like progenitor through M. avium—M. gallinarum and to a proposal for the polyvalent vaccine currently being developed by this research group.     

Mycobacterium Species Related to M. leprae and M. lepromatosis from Cows with Bovine Nodular Thelitis

Bovine nodular thelitis is a granulomatous dermatitis associated with infection with acid-fast bacteria. To identify the mycobacterium responsible for this infection, we conducted phylogenetic investigations based on partial sequencing of 6 genes. These bacteria were identified as an undescribed Mycobacterium species that was phylogenetically related to M. leprae and M. lepromatosis.     

Immunization against leprosy: progress and prospects

The limitations of the current approach to leprosy control through mass treatment of patients are well recognized. The long incubation period of the disease, the insidious onset, the chronic course, and the need for prolonged treatment have made control a formidable task. The recent years have seen tremendous progress in the field of immunology of leprosy, and the availability of large quantities of Mycobacterium leprae, grown in the nine-banded armadillo, has given impetus to the search for a vaccine specific for leprosy. Methods for production and purification of M. leprae have now been developed and the resulting preparation has been shown to produce good delayed-type hypersensitivity in mice and guinea pigs.