Evaluation of qPCR-Based Assays for Leprosy Diagnosis Directly in Clinical Specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations

PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

Made available in DSpace on 2015-05-22T18:42:45Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) alejandra_martinezetal_IOC_2014.pdf: 211978 bytes, checksum: 016e6ec5b676ea1547991d9506ce858f (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil / Tulane National Primate Research Center. Divisions of Bacteriology and Parasitology. Louisiania, USA.Fundação Alfredo da Matta. Manaus, AM, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Universidade Nilton Lins. Manaus, AM, Brasil.In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis

Selected results obtained by PCR assays tested in frozen and fresh skin biopsies from leprosy patients.

<p>Selected results obtained by PCR assays tested in frozen and fresh skin biopsies from leprosy patients.</p

Selected data showing PCR assays tested in nasal swabs or blood from healthy individuals and household contacts.

<p>*household contacts with multibacillary patients as index case (IC).</p

Detection of Mycobacterium leprae DNA by Polymerase Chain Reaction in the Blood and Nasal Secretion of Brazilian Household Contacts

Submitted by Sandra Infurna ([email protected]) on 2019-05-30T11:38:41Z No. of bitstreams: 1 EuzenirSarno_MiltonMoraes_etal_IOC_2004.pdf: 26414 bytes, checksum: c8d8685442efc65a4cb5480a0bd9378a (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-05-30T11:43:58Z (GMT) No. of bitstreams: 1 EuzenirSarno_MiltonMoraes_etal_IOC_2004.pdf: 26414 bytes, checksum: c8d8685442efc65a4cb5480a0bd9378a (MD5)Made available in DSpace on 2019-05-30T11:43:58Z (GMT). No. of bitstreams: 1 EuzenirSarno_MiltonMoraes_etal_IOC_2004.pdf: 26414 bytes, checksum: c8d8685442efc65a4cb5480a0bd9378a (MD5) Previous issue date: 2004Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Medicina Tropical. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Medicina Tropical. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Medicina Tropical. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Medicina Tropical. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Medicina Tropical. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Medicina Tropical. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Medicina Tropical. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Medicina Tropical. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.DNA samples from blood and nasal swabs of 125 healthy household contacts was submitted to amplification by polymerase chain reaction (PCR) using a Mycobacterium leprae-specific sequence as a target for the detection of subclinical infection with M. leprae. All samples were submitted to hybridization analysis in order to exclude any false positive or negative results. Two positive samples were confirmed from blood out of 119 (1.7%) and two positive samples from nasal secretion out of 120 (1.7%). The analysis of the families with positive individuals showed that 2.5% (n = 3) of the contacts were relatives of multibacilary patients while 0.8% of the cases (n = 1) had a paucibacilary as an index case. All positive contacts were followed up and after one year none of them presented clinical signs of the disease. In spite of the PCR sensitivity to detect the presence of the M. leprae in a subclinical stage, this molecular approach did not seem to be a valuable tool to screen household contacts, since we determined a spurious association of the PCR positivity and further development of leprosy

SHORT COMMUNICATION - Detection of Mycobacterium leprae DNA by Polymerase Chain Reaction in the Blood and Nasal Secretion of Brazilian Household Contacts

DNA samples from blood and nasal swabs of 125 healthy household contacts was submitted to amplification by polymerase chain reaction (PCR) using a Mycobacterium leprae-specific sequence as a target for the detection of subclinical infection with M. leprae. All samples were submitted to hybridization analysis in order to exclude any false positive or negative results. Two positive samples were confirmed from blood out of 119 (1.7%) and two positive samples from nasal secretion out of 120 (1.7%). The analysis of the families with positive individuals showed that 2.5% (n = 3) of the contacts were relatives of multibacilary patients while 0.8% of the cases (n = 1) had a paucibacilary as an index case. All positive contacts were followed up and after one year none of them presented clinical signs of the disease. In spite of the PCR sensitivity to detect the presence of the M. leprae in a subclinical stage, this molecular approach did not seem to be a valuable tool to screen household contacts, since we determined a spurious association of the PCR positivity and further development of leprosy

Genetic diversity of Mycobacterium leprae isolates from Brazilian leprosy patients

Submitted by Sandra Infurna ([email protected]) on 2018-09-25T17:17:57Z No. of bitstreams: 1 amanda_fontes_etal_IOC_2009.pdf: 376703 bytes, checksum: ec0c4b7b9b14df59fc4f776733a26280 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-09-25T17:24:17Z (GMT) No. of bitstreams: 1 amanda_fontes_etal_IOC_2009.pdf: 376703 bytes, checksum: ec0c4b7b9b14df59fc4f776733a26280 (MD5)Made available in DSpace on 2018-09-25T17:24:17Z (GMT). No. of bitstreams: 1 amanda_fontes_etal_IOC_2009.pdf: 376703 bytes, checksum: ec0c4b7b9b14df59fc4f776733a26280 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ. Brasil.Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, Colorado, USA.Instituto Lauro de Souza Lima, Bauru. SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, Colorado, USA.Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, Colorado, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ. Brasil.Introduction Leprosy is a chronic disease caused by infection with Mycobacterium leprae, an obligate intracellular parasite. A problem in studying the transmission of leprosy is the small amount of variation in bacterial genomic DNA. The discovery of variable number of tandem repeats (VNTRs) allowed the detection of strain variation in areas with a high prevalence of leprosy. Four genotypes of M. leprae based on three single-nucleotide polymorphism (SNPs) were also discovered to be useful for analysis of the global spread of leprosy