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Use of the polymerase chain reaction to detect Mycobacterium leprae in urine
Caleffi, K.R.;Hirata, R.D.C.;Hirata, M.H.;Caleffi, E.R.;Siqueira, V.L.D.;Cardoso, R.F.;
Brazilian Journal of Medical and Biological Research , 2012, DOI: 10.1590/S0100-879X2012007500011
Abstract: leprosy is an infectious disease caused by mycobacterium leprae. the polymerase chain reaction (pcr) has been applied to detect m. leprae in different clinical samples and urine seems to be attractive for this purpose. pcr was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp pcr fragment of the m. leprae pra gene (pcr-pra) in urine samples. seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in maringá, pr, brazil. of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (tt) and dapsone, rifampicin and clofazimine for borderline (bb) and lepromatous (ll) forms. the control group contained 50 healthy individuals without any clinical history of leprosy. dna isolated from leprosy patients’ urine samples was successfully amplified by pcr-pra in 46.6% (34/73) of the cases. the positivity of pcr-pra for patients with the tt form was 75% for both patients under treatment and non-treated patients (p = 0.1306). in patients with the ll form, pcr-pra positivity was 52 and 30% for patients under treatment and non-treated patients, respectively (p = 0.2386). pcr-pra showed a statistically significant difference in detecting m. leprae between the tt and ll forms of leprosy in patients under treatment (p = 0.0033). although the current study showed that the proposed pcr-pra has some limitations in the detection of m. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in tt leprosy where the afb slit-skin smear is always negative.
Pathogen-Specific Epitopes as Epidemiological Tools for Defining the Magnitude of Mycobacterium leprae Transmission in Areas Endemic for Leprosy  [PDF]
Marcia V. S. B. Martins equal contributor,Marjorie M. da S. Guimar?es equal contributor,John S. Spencer,Mariana A. V. B. Hacker,Luciana S. Costa,Fernanda M. Carvalho,Annemieke Geluk,Jolien J. van der Ploeg-van Schip,Maria A. A. Pontes,Heitor S. Gon?alves,Janvier P. de Morais,Tereza J. P. G. Bandeira,Maria C. V. Pessolani,Patrick J. Brennan,Geraldo M. B. Pereira
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001616
Abstract: During recent years, comparative genomic analysis has allowed the identification of Mycobacterium leprae-specific genes with potential application for the diagnosis of leprosy. In a previous study, 58 synthetic peptides derived from these sequences were tested for their ability to induce production of IFN-γ in PBMC from endemic controls (EC) with unknown exposure to M. leprae, household contacts of leprosy patients and patients, indicating the potential of these synthetic peptides for the diagnosis of sub- or preclinical forms of leprosy. In the present study, the patterns of IFN-γ release of the individuals exposed or non-exposed to M. leprae were compared using an Artificial Neural Network algorithm, and the most promising M. leprae peptides for the identification of exposed people were selected. This subset of M. leprae-specific peptides allowed the differentiation of groups of individuals from sites hyperendemic for leprosy versus those from areas with lower level detection rates. A progressive reduction in the IFN-γ levels in response to the peptides was seen when contacts of multibacillary (MB) patients were compared to other less exposed groups, suggesting a down modulation of IFN-γ production with an increase in bacillary load or exposure to M. leprae. The data generated indicate that an IFN-γ assay based on these peptides applied individually or as a pool can be used as a new tool for predicting the magnitude of M. leprae transmission in a given population.
Deciphering the contribution of lipid droplets in leprosy: multifunctional organelles with roles in Mycobacterium leprae pathogenesis
Mattos, Katherine Antunes de;Sarno, Euzenir Nunes;Pessolani, Maria Cristina Vidal;Bozza, Patricia T;
Memórias do Instituto Oswaldo Cruz , 2012, DOI: 10.1590/S0074-02762012000900023
Abstract: leprosy is an infectious disease caused by mycobacterium leprae that affects the skin and nerves, presenting a singular clinical picture. across the leprosy spectrum, lepromatous leprosy (ll) exhibits a classical hallmark: the presence of a collection of m. leprae-infected foamy macrophages/schwann cells characterised by their high lipid content. the significance of this foamy aspect in mycobacterial infections has garnered renewed attention in leprosy due to the recent observation that the foamy aspect represents cells enriched in lipid droplets (ld) (also known as lipid bodies). here, we discuss the contemporary view of ld as highly regulated organelles with key functions in m. leprae persistence in the ll end of the spectrum. the modern methods of studying this ancient disease have contributed to recent findings that describe m. leprae-triggered ld biogenesis and recruitment as effective mycobacterial intracellular strategies for acquiring lipids, sheltering and/or dampening the immune response and favouring bacterial survival, likely representing a fundamental aspect of m. leprae pathogenesis. the multifaceted functions attributed to the ld in leprosy may contribute to the development of new strategies for adjunctive anti-leprosy therapies.
Peptides Derived from Mycobacterium leprae ML1601c Discriminate between Leprosy Patients and Healthy Endemic Controls  [PDF]
Kidist Bobosha,Jolien J. van der Ploeg-van Schip,Danuza A. Esquenazi,Marjorie M. Guimar?es,Marcia V. Martins,Yonas Bekele,Yonas Fantahun,Abraham Aseffa,Kees L. M. C. Franken,Ronaldo C. Gismondi,Maria C. V. Pessolani,Tom H. M. Ottenhoff,Geraldo M. B. Pereira,Annemieke Geluk
Journal of Tropical Medicine , 2012, DOI: 10.1155/2012/132049
Abstract: The stable incidence of new leprosy cases suggests that transmission of infection continues despite worldwide implementation of MDT. Thus, specific tools are needed to diagnose early stage Mycobacterium leprae infection, the likely sources of transmission. M. leprae antigens that induce T-cell responses in M. leprae exposed and/or infected individuals thus are major targets for new diagnostic tools. Previously, we showed that ML1601c was immunogenic in patients and healthy household contacts (HHC). However, some endemic controls (EC) also recognized this protein. To improve the diagnostic potential, IFN-γ responses to ML1601c peptides were assessed using PBMC from Brazilian leprosy patients and EC. Five ML1601c peptides only induced IFN-γ in patients and HHC. Moreover, 24-hour whole-blood assay (WBA), two ML1601c peptides could assess the level of M. leprae exposure in Ethiopian EC. Beside IFN-γ, also IP-10, IL-6, IL-1β, TNF-α, and MCP-1 were increased in EC from areas with high leprosy prevalence in response to these ML1601c peptides. Thus, ML1601c peptides may be useful for differentiating M. leprae exposed or infected individuals and can also be used to indicate the magnitude of M. leprae transmission even in the context of various HLA alleles as present in these different genetic backgrounds. 1. Introduction Leprosy is a treatable infection caused by Mycobacterium leprae (M. leprae) involving skin and peripheral nerves and is influenced by genetic and environmental factors [1–3]. The infection can result in skin lesions, nerve degeneration, and deformities. Despite a spectacular decrease in global prevalence since 1982, transmission of leprosy is sustained as evidenced by the hundreds of thousand new cases of leprosy that keep being detected globally every year: 244,796 new cases of leprosy were detected during 2009 amongst whom 22,485 were children and the registered prevalence at the beginning of 2010 was 211,903 cases [4]. In Brazil, for example, the number of new cases detected during 2009 was 37,610 resulting in a registered prevalence of 38,179 at the end of first quarter of 2010 [4]. These figures demonstrate that M. leprae-infected contacts and persons with subclinical, undiagnosed leprosy, likely the major sources of unidentified transmission, are an incessant source of active transmission. Despite many efforts, prediction of disease development in affected individuals is still not possible nor can we detect asymptomatic M. leprae infection. Diagnosis of leprosy is usually based on clinical features and skin smear results including the
Infection by Mycobacterium leprae of household contacts of lepromatous leprosy patients from a post-elimination leprosy region of Colombia
Cardona-Castro, Nora M;Restrepo-Jaramillo, Sandra;Gil de la Ossa, Myriam;Brennan, Patrick J;
Memórias do Instituto Oswaldo Cruz , 2005, DOI: 10.1590/S0074-02762005000700003
Abstract: the leprosy control program of antioquia, (post-elimination leprosy state of colombia), had registered by 1999, 56 lepromatous leprosy patients and their household contacts (hhc). our interest was to detect mycobacterium leprae infection in these hhc. clinical examination, acid-fast bacillary staining (afb) in nasal secretions, and slit skin samples, igm anti-pgl-i in serum and lepromine a (mitsuda) reactivity were tested. two hundred forty eight hhc were studied, 49% were male. after clinical examination, two hhc were diagnosed as multi bacillary patients; 13% showed positive igm anti-pgl-i titers; mitsuda reaction (> 4 mm) was positive in 59%; afb was negative in all samples, except in the two new patients. hhc were classified according to test results.group 1: two new multi bacillary patients. group 2: 15 hhc seropositive, mitsuda-negative. group 3: 13 hhc seropositive, mitsuda-positive. group 4: 130 hhc seronegative, mitsuda-positive. group 5: 88 hhc seronegative, mitsuda-negative. these results are an indication that the transmission of the infection is still happening in a region considered in the post elimination phase. the two new patients represent an infection source for others contacts, and groups 2 and 3 are infected hhc that could develop the disease in future. follow up of high risk population is necessary to achieve real elimination of leprosy.
Mycobacterium leprae in the periodontium, saliva and skin smears of leprosy patients
Abdalla, Ligia Fernandes;Santos, Jo?o Hugo Abdalla;Collado, Carolina Souza Cunha;Cunha, Maria da Gra?a Souza;Naveca, Felipe Gomes;
Revista Odonto Ciência , 2010, DOI: 10.1590/S1980-65232010000200008
Abstract: purpose: to verify the presence of m. leprae in the periodontium, saliva and skin slit smears of leprosy patients. to correlate bacteriological and molecular findings with clinical data and compare laboratory techniques. methods: a cross-sectional study was designed to use bacteriological (baciloscopy) and molecular (pcr) parameters to detect m. leprae in exudates of the gingival sulcus/periodontium pocket, saliva and skin slit smears from multiple clinical forms of leprosy patients without previous treatment. results: the study included 48 leprosy patients with 15 multibacillary (mb) cases and 33 paucibacillary (pb) cases. the diagnosis of mb was confirmed through bacteriological examination and pcr results from skin slit smears. a total of 16 (48.5%) pb patients were pcr positive only. four pb patients with negative pcr skin smears were pcr positive for the periodontium and saliva, with 2 cases and 1 case, respectively. no periodontium or saliva samples had positive bacteriological results. conclusion: there was no correlation between periodontal disease and the presence of m. leprae. bacteriological examination did not prove to be an efficient technique for the analysis of saliva and periodontium samples. pcr analysis of skin smears was more efficient at diagnosing pb patients than bacteriological examination. pcr positive results for the detection of m. leprae in pb patients can be increased by collecting slit skin smears, periodontium and saliva samples.
Survey to identify Mycobacterium leprae-infected household contacts of patients from prevalent regions of leprosy in Colombia
Cardona-Castro, N;Beltrán-Alzate, JC;Manrique-Hernández, R;
Memórias do Instituto Oswaldo Cruz , 2008, DOI: 10.1590/S0074-02762008000400003
Abstract: leprosy in colombia is in the post-elimination phase; nevertheless, there are regions of this country where the incidence is still around 3-4/100,000. early detection of leprosy patients is a priority for achieving control and elimination of leprosy; however, the clinical exam is not very sensitive and thus, the majority of patients are diagnosed only when they demonstrate lesions, and damage to the nerves and skin has already occurred. the goal of the present study was to identify mycobacterium leprae infection and immune responses in household contacts (hhc) of leprosy patients from three prevalent regions of colombia. clinical examination, the mitsuda test, evaluation of igm anti-pgl-i in the serum, the bacillar index (bi), and polymerase chain reaction (pcr) from nasal swabs (ns) were performed for 402 hhc of 104 leprosy patients during a cross-sectional survey. positive titers for igm anti-pgl1 were found for 54 hhc, and pcr-positive ns for 22. the mitsuda reaction was negative for 38 hhc, although three were positive for igm anti-pgl-1 titers. the data document that leprosy transmission among hhc is still occurring in a non-endemic country.
Seroreactivity to new Mycobacterium leprae protein antigens in different leprosy-endemic regions in Brazil
Hungria, Emerith Mayra;Oliveira, Regiane Morillas de;Souza, Ana Lúcia Osório Maroclo de;Costa, Maurício Barcelos;Souza, Vania Nieto Brito de;Silva, Eliane Aparecida;Moreno, Fátima Regina Vilani;Nogueira, Maria Esther Salles;Costa, Maria Renata Sales Nogueira;Silva, S?nia Maria Usó Ruiz;Bührer-Sékula, Samira;Reed, Steven G;Duthie, Malcolm S;Stefani, Mariane Martins de Araújo;
Memórias do Instituto Oswaldo Cruz , 2012, DOI: 10.1590/S0074-02762012000900017
Abstract: new mycobacterium leprae protein antigens can contribute to improved serologic tests for leprosy diagnosis/classification and multidrug therapy (mdt) monitoring. this study describes seroreactivity to m. leprae proteins among participants from three highly endemic leprosy areas in brazil: central-western goiania/goiás (go) (n = 225), rondonópolis/mato grosso (mt) (n = 764) and northern prata village/pará (pa) (n = 93). elisa was performed to detect igg to proteins (92f, 46f, leprosy idri diagnostic-1, ml0405, ml1213) and igm to phenolic glycolipid-i (pgl-i). multibacillary (mb) leprosy had positive rates for pgl-i that were similar to those for proteins; however, some anti-pgl-i-negative subjects were positive for proteins, suggesting that adding protein antigen to pgl-i can enhance the sensitivity of mb leprosy detection. in mt, different degrees of seroreactivity were observed and ranked for mb, former patients after mdt, paucibacillary (pb) leprosy, household contact (hhc) and endemic control (ec) groups. the seroreactivity of pb patients was low in go and mt. hhcs from different endemic sites had similar igg antibody responses to proteins. 46f and 92f were not recognised by most tuberculosis patients, ecs or hhcs within go, an area with high bcg vaccination coverage. low positivity in ec and hhc was observed in pa and mt. our results provide evidence for the development of an improved serologic test that could be widely applicable for mb leprosy testing in brazil.
Variation of TTC Repeat Pattern In The Dna of Mycobacterium Leprae Isolates Obtained from Archeological Bones and Leprosy Patients From East Nusa Tenggara  [PDF]
Dinar Adriaty,Ratna Wahyuni,Iswahyudi,Bimo Aksono
Journal of Tropical Life Science , 2012,
Abstract: The existence of leprosy or kusta or Morbus Hansen or Hansen’s disease has been known for years, including in Indonesia. Starting from the discovery of Mycobacterium leprae isolates from ancient bone (about 1.000 years B.C), the archaeological excavations results in East Nusa Tenggara, interesting questions arise about how the development of leprosy in eastern Indonesia is. Biology molecular study would become a powerful tool to investigate the presence of leprosy bacillary whether there are similarities between the genomes of M. leprae isolates in the primeval and the present. PCR examinations were performed on mandibular bone fragments from ancient human who lived 1000 years B.C. discovered in archaeological surveys on the island of Lembata and three leprosy patients from East Nusa Tenggara. The DNA extraction was performed using a kit from Qiagen products and its TTC repeating pattern was seen with the method of direct sequencing. It turned out that the TTC profile obtained from samples of archaeological was as many as 13 copies, while the repetition of TTC in three samples of leprosy patients were 15, 17 and 26 copies. The different number of TTC repetition shows the different isolates of M. leprae between in the ancient times and the present. Further studies are needed to verify the differences in the genome that occur, for example from the study of SNPs (single nucleotide polymorphisms).
Use of Short Tandem Repeat Sequences to Study Mycobacterium leprae in Leprosy Patients in Malawi and India  [PDF]
Saroj K. Young,Jorg M. Ponnighaus,Suman Jain,Sebastian Lucas,Sujai Suneetha,Diana N. J. Lockwood,Douglas B. Young,Paul E. M. Fine
PLOS Neglected Tropical Diseases , 2008, DOI: 10.1371/journal.pntd.0000214
Abstract: Background Inadequate understanding of the transmission of Mycobacterium leprae makes it difficult to predict the impact of leprosy control interventions. Genotypic tests that allow tracking of individual bacterial strains would strengthen epidemiological studies and contribute to our understanding of the disease. Methodology/Principal Findings Genotyping assays based on variation in the copy number of short tandem repeat sequences were applied to biopsies collected in population-based epidemiological studies of leprosy in northern Malawi, and from members of multi-case households in Hyderabad, India. In the Malawi series, considerable genotypic variability was observed between patients, and also within patients, when isolates were collected at different times or from different tissues. Less within-patient variability was observed when isolates were collected from similar tissues at the same time. Less genotypic variability was noted amongst the closely related Indian patients than in the Malawi series. Conclusions/Significance Lineages of M. leprae undergo changes in their pattern of short tandem repeat sequences over time. Genetic divergence is particularly likely between bacilli inhabiting different (e.g., skin and nerve) tissues. Such variability makes short tandem repeat sequences unsuitable as a general tool for population-based strain typing of M. leprae, or for distinguishing relapse from reinfection. Careful use of these markers may provide insights into the development of disease within individuals and for tracking of short transmission chains.
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