BackgroundMycobacterium leprae transcriptomic and human host immune gene expression signatures that demonstrate a plausible association with type I (T1R) and type II reactions (T2R) aid in early... Show moreBackgroundMycobacterium leprae transcriptomic and human host immune gene expression signatures that demonstrate a plausible association with type I (T1R) and type II reactions (T2R) aid in early diagnosis, prevention of nerve damage and consequent demyelinating neuropathy in leprosy. The aim of the study is to identify M. leprae and host-associated gene-expression signatures that are associated with reactional states in leprosy. MethodsThe differentially expressed genes from the whole transcriptome of M. leprae were determined using genome-wide hybridization arrays with RNA extracted from skin biopsies of 20 T1R, 20 T2R and 20 non reactional controls (NR). Additionally, human immune gene-expressions were profiled using RT2-PCR profiler arrays and real-time qPCRs. ResultsThe RNA quality was optimal in 16 NR, 18 T1R and 19 T2R samples. Whole transcriptome expression array of these samples revealed significant upregulation of the genes that encode integral and intrinsic membrane proteins, hydrolases and oxidoreductases. In T1R lesional skin biopsy specimens, the top 10 significantly upregulated genes are ML2064, ML1271, ML1960, ML1220, ML2498, ML1996, ML2388, ML0429, ML2030 and ML0224 in comparison to NR. In T2R, genes ML2498, ML1526, ML0394, ML1960, ML2388, ML0429, ML0281, ML1847, ML1618 and ML1271 were significantly upregulated. We noted ML2664 was significantly upregulated in T1R and repressed in T2R. Conversely, we have not noted any genes upregulated in T2R and repressed in T1R. In both T1R and T2R, ML2388 was significantly upregulated. This gene encodes a probable membrane protein and epitope prediction using Bepipred-2.0 revealed a distinct B-cell epitope. Overexpression of ML2388 was noted consistently across the reaction samples. From the host immune gene expression profiles, genes for CXCL9, CXCL10, CXCL2, CD40LG, IL17A and CXCL11 were upregulated in T1R when compared to the NR. In T2R, CXCL10, CXCL11, CXCL9, CXCL2 and CD40LG were upregulated when compared to the NR group. ConclusionA gene set signature involving bacterial genes ML2388, ML2664, and host immune genes CXCL10 and IL-17A can be transcriptomic markers for reactional states in leprosy. Show less
Leprosy is an infectious disease that affects peripheral nerves and can lead to severe lifelong disabilities. Despite the availability of an effective cure, a fairly stable number of about 200,000... Show moreLeprosy is an infectious disease that affects peripheral nerves and can lead to severe lifelong disabilities. Despite the availability of an effective cure, a fairly stable number of about 200,000 new leprosy patients per year has been reported since 2010. This stagnation shows that the transmission of the mycobacteria that cause leprosy, Mycobacterium leprae and Mycobacterium lepromatosis, is still taking place. Timely diagnosis of leprosy patients is therefore vital, so that the time frame in which a person is contagious is shortened, but also irreversible nerve damage and leprosy-associated disabilities can be prevented. However, tools that confirm the diagnosis of leprosy are not yet available. This thesis investigated which factors in blood (the so-called biomarkers) can help to diagnose leprosy. The clinical signs of leprosy have a spectral character and are influenced by the immune response of the host. A combination of biomarkers is described that is able to identify patients with a lot of bacteria (multibacillary) as well as the more difficult to diagnose patients with few bacteria (paucibacillary). Subsequently, these biomarkers have been implemented in user-friendly lateral flow assays, which have been extensively validated in leprosy endemic areas. Show less
Leprosy is a multifactorial chronic disease caused by Mycobacterium leprae or Mycobacterium lepromatosis that affects the skin and nerves. More than 200.000 new cases are diagnosed per year; thus,... Show moreLeprosy is a multifactorial chronic disease caused by Mycobacterium leprae or Mycobacterium lepromatosis that affects the skin and nerves. More than 200.000 new cases are diagnosed per year; thus, transmission is still ongoing. The most likely way of transmission is the respiratory route form human-to-human; however, transmission is still not clearly understood. Early diagnosis of leprosy is crucial to reduce and avoid transmission as well as leprosy-associated disabilities, which are also a cause of stigma. Currently, diagnosis is performed based on clinical signs and symptoms and late- or mis-diagnosis are not uncommon.In this thesis, we combined the study of pathogen transmission with host transcriptomic and genomic biomarkers. To explore M. leprae transmission a One Health approach was followed, where human, animal and environmental samples were studied.The combination of demographic characteristics, pathogen detection, genetic and/or transcriptomic biomarkers can be applied in a multifactorial leprosy signature applicable for early diagnosis of leprosy and/or to guide intervention strategies. Identification of predictive biomarkers will in due course lead to prompt treatment, preventing leprosy-associated irreversible disabilities as well as reducing M. leprae transmission. Show less
Mycobacterium leprae, the causative agent of leprosy, is still actively transmitted in endemic areas reflected by the fairly stable number of new cases detected each year. Recognizing the signs and... Show moreMycobacterium leprae, the causative agent of leprosy, is still actively transmitted in endemic areas reflected by the fairly stable number of new cases detected each year. Recognizing the signs and symptoms of leprosy is challenging, especially at an early stage. Improved diagnostic tools, based on sensitive and specific biomarkers, that facilitate diagnosis of leprosy are therefore urgently needed. In this review, we address the challenges that leprosy biomarker research is facing by reviewing cell types reported to be involved in host immunity to M leprae. These cell types can be associated with different possible fates of M leprae infection being either protective immunity, or pathogenic immune responses inducing nerve damage. Unraveling these responses will facilitate the search for biomarkers. Implications for further studies to disentangle the complex interplay between host responses that lead to leprosy disease are discussed, providing leads for the identification of new biomarkers to improve leprosy diagnostics. Show less
Human settlement of Madagascar traces back to the beginning of the first millennium with the arrival of Austronesians from Southeast Asia, followed by migrations from Africa and the Middle East.... Show moreHuman settlement of Madagascar traces back to the beginning of the first millennium with the arrival of Austronesians from Southeast Asia, followed by migrations from Africa and the Middle East. Remains of these different cultural, genetic, and linguistic legacies are still present in Madagascar and other islands of the Indian Ocean. The close relationship between human migration and the introduction and spread of infectious diseases, a well-documented phenomenon, is particularly evident for the causative agent of leprosy, Mycobacterium leprae. In this study, we used whole-genome sequencing (WGS) and molecular dating to characterize the genetic background and retrace the origin of the M. leprae strains circulating in Madagascar (n = 30) and the Comoros (n = 3), two islands where leprosy is still considered a public health problem and monitored as part of a drug resistance surveillance program. Most M. leprae strains (97%) from Madagascar and Comoros belonged to a new genotype as part of branch 1, closely related to single nucleotide polymorphism (SNP) type 1D, named 1D-Malagasy. Other strains belonged to the genotype 1A (3%). We sequenced 39 strains from nine other countries, which, together with previously published genomes, amounted to 242 genomes that were used for molecular dating. Specific SNP markers for the new 1D-Malagasy genotype were used to screen samples from 11 countries and revealed this genotype to be restricted to Madagascar, with the sole exception being a strain from Malawi. The overall analysis thus ruled out a possible introduction of leprosy by the Austronesian settlers and suggests a later origin from East Africa, the Middle East, or South Asia. Show less
Globally more than 200,000 people develop leprosy every year and 2-3 million people live with leprosy associated disabilities. Despite the availability of multi drug therapy, leprosy has continued... Show moreGlobally more than 200,000 people develop leprosy every year and 2-3 million people live with leprosy associated disabilities. Despite the availability of multi drug therapy, leprosy has continued affecting many individuals, including children because of the uninterrupted transmission in the population. Untreated multi bacillary cases as well as non-symptomatic M. leprae infected individuals in the population are believed to be the major sources of M. leprae infection and transmission. Leprosy reactions are also the major causes of disabilities. However, no tools are available to predict their occurrence. This thesis focuses on in vitro assessment of recombinant M. leprae proteins and synthetic peptides for their immunogenicity and specificity in populations with different genetic backgrounds by measuring cell mediated immunity and this has shown the presence of potential antigens. Further in depth analysis of the host immune responses against these unique antigens in leprosy patients, their household contacts and healthy endemic controls has led to identification of potential biomarkers with an immense importance in development of diagnostic tools for detection of M. leprae infection and early diagnosis of leprosy reactions. Currently, field friendly tests for early detection are developed at the LUMC using identified M. leprae antigens and host biomarkers with diagnostic potential. Show less
