qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients

Araújo, Sérgio; Goulart, Luíz Ricardo; Truman, Richard W.; Goulart, Isabela Maria Bernardes; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masao; Suffys, Philip Noël; Fontes, Amanda B.; Rosa, Patrícia Sammarco; Scollard, David M.; Williams, Diana L.

doi:10.1371/journal.pntd.0005506

Cited by 17 publications

(12 citation statements)

References 19 publications

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“…Confirmation of mixed alleles in the folP1, gyrA, and rpoB genes of M. leprae has been reported previously. 23,24 Mutation V48A has been detected by Nakata et al in their clinical samples. 25 In this study, we analyze V48A mutant using in silico method.…”

Section: Discussionmentioning

confidence: 92%

In silico analysis of V48A dihydropteroate synthase mutation to dapsone on Mycobacterium leprae from Papua

Maladan¹,

Krismawati²,

Hutapea³

et al. 2020

hsji

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Latar belakang: Lepra merupakan penyakit yang disebabkan oleh Mycobacterium leprae. Resistensi obat merupakan salah satu tantangan dalam pemberantasan kusta khususnya di Papua. Adanya mutasi pada gen folP1 penyandi dihydropteroate synthase (DHPS) merupakan dasar untuk deteksi molekuler resistensi dapson pada penyakit lepra. Tujuan penelitian ini adalah mendeteksi mutasi pada gen folP1 Mycobacterium leprae dari Papua, Indonesia dan menganalisis pengaruh mutasi tersebut terhadap dapson dengan metode in silico. Metode: Identifikasi mutasi pada gen folp1 M. leprae dilakukan melalui proses Basic Local Alignment Search Tool (BLAST) di gene bank. Analisis efek mutasi dengan menggunakan server Have (y) Our Protein Explained (HOPE). Prediksi binding pocket menggunakan Computed Atlas of Surface Topography of proteins (CASTp). Homologi modeling struktur 3D DHPS menggunakan server Iterative Threading ASSEmbly Refinement (I TASSER). Analisis docking dengan menggunakan AutoDock Vina yang terintegrasi dengan aplikasi Python Prescription (PyRx). Hasil: Hasil sekuensing menunjukkan adanya variasi dalam gen folP1 M. leprae yaitu perubahan dari Timin (T) menjadi Sitosin (C) pada nukleotida 143. Residu yang bermutasi (V48A) terletak pada domain yang penting untuk aktivitas protein dan kontak dengan residu di domain lain. Ada kemungkinan bahwa interaksi ini penting untuk fungsi protein secara benar. Mutan V48A tidak banyak mempengaruhi stabilitas dari dihydropteroate synthase M. leprae. Kesimpulan: Berdasarkan analisis molecular docking, mutasi V48A tidak mempengaruhi binding affinity dapson terhadap dihydropteroate synthase M. leprae. Hasil ini menunjukkan mutan V48A kemungkinan tetaprentan terhadap dapson. Dengan demikian perlu dilakukan uji in vivo untuk mengkofirmasi efek mutasi V48A. Kata kunci: Mycobacterium leprae, folP1 gene, dihydropteroate synthase, dapson Abstract Background: Leprosy is a disease caused by Mycobacterium leprae. Drug resistance is one of the challenges in leprosy elimination especially in Papua. The presence of mutations in folP1 gene that encode dihydropteroate synthase (DHPS) was considered as the exclusive basis for molecular detection of dapsone resistance in leprosy. The objective of this study was to detect mutations in the folP1 gene of Mycobacterium leprae from Papua, Indonesia and to analyze the effect of these mutations on dapsone using the in-silico method. Methods: Identification of mutations in the folp1 M. leprae gene is carried out through the Basic Local Alignment Search Tool (BLAST) process in the gene bank. The analysis of the effects of mutations using the Have (y)Our Protein Explained (HOPE) server. Bindings pocket prediction is done using the Computed Atlas of Surface Topography of proteins (CASTp). Homology modeling 3D structure of DHPS using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Docking analysis was performed using AutoDock Vina which is integrated with the Python Prescription (PyRx) application. Results: The sequencing results showed a variation in the folP1 M. leprae gene, namely a change from thymine (T) to cytosine (C) in nucleotide 143. The mutated residue (V48A) is in a domain that is essential for the activity of the protein and in contact with residues in another domain. It is possible that this interaction is important for the correct function of the protein. V48A mutants did not significantly affect the stability of DHPS M. leprae. Conclusion: Based on molecular docking analysis, this mutation does not affect binding affinity dapsone against M. leprae dihydropteroate synthase. These results indicate that the V48A mutant is likely to remain susceptible to dapsone. Thus, it is necessary to do an in vivo test to confirm the effect of the V48A mutation. Keywords: Mycobacterium leprae, folP1 gene, dihydropteroate synthase, dapsone

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Section: Discussionmentioning

confidence: 92%

In silico analysis of V48A dihydropteroate synthase mutation to dapsone on Mycobacterium leprae from Papua

Maladan¹,

Krismawati²,

Hutapea³

et al. 2020

hsji

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“…Based on the result of an antimicrobial resistance study on leprosy by the WHO surveillance network, skin biopsy specimens from MB leprosy cases at sentinel sites of 19 countries from the period 2009–2015 were included, and resistance to rifampicin, dapsone and ofloxacin according to PCR sequencing of rpoB, folP1 and gyrA gene DRDRs was studied [21]. The PCR/sequencing method has been widely applied in MB leprosy patients; however, drug resistance among PB leprosy patients and the use of FFPE specimens have seldom been discussed [15]. In this study, we developed a nested PCR and TaqMan SNP Genotyping Assay, which were used for drug resistance testing in MB and PB patients.…”

Section: Discussionmentioning

confidence: 99%

“…Testing methods include M . leprae DRDR primers and a polymerase chain reaction (PCR) sequencing method recommended by the WHO [13], GenoType LepraeDR [14], and qPCR-high-resolution melt analysis [15], among others. However, the use of nested PCR and the TaqMan SNP Genotyping Assay for this type of testing has not been reported.…”

Section: Introductionmentioning

confidence: 99%

Nested PCR and the TaqMan SNP Genotyping Assay enhanced the sensitivity of drug resistance testing of Mycobacterium leprae using clinical specimens of leprosy patients

Chen

Liu

et al. 2019

PLoS Negl Trop Dis

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BackgroundAlthough leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China.Methodology/Principal findingsSeventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94–86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes.Conclusions/SignificanceNested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.

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“…A significant advance in increasing bacillus identification occurred with the use of real-time PCR technology. This methodology has been used in the follow-up of leprosy patients undergoing treatment [63] evaluation of bacterial load [13] viable bacterial load [60] and determination of resistance to treatment [14].…”

Section: Complementary Molecular Testsmentioning

confidence: 99%

“…The use of phenol-glycolipid-1 (PGL-1) [9], lipoarabinomannan (LAM) [1] and heat shock proteins (GroES and GroEL) [10] as antigens for the enzyme-linked immunosorbent assay (ELISA) and in immunosensors can be validated as methods for detecting new cases of the disease and for early diagnosis [11]. In addition, molecular tests aid in the identification of specific M. leprae sequences in clinical samples, which can be amplified through the polymerase chain reaction (PCR) technique, allowing DNA detection of the infectious agent [12] and/or through the use of real-time PCR technology that allows the evaluation of bacterial load [13] and also the monitoring of drug resistance [14].…”

Section: Introductionmentioning

confidence: 99%

Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

Lima¹,

Moraes²,

Pinho³

et al. 2019

Hansen's Disease - The Forgotten and Neglected Disease

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Leprosy is a worldwide health problem, which needs the development of new and innovative strategies to be controlled. Early diagnosis of leprosy is an important contribution to reducing the incidence of the disease; thus, the development of biotechnology platforms, which include the mapping of antigens with potential to be used in immunodiagnostic and molecular methods for the detection of Mycobacterium leprae, is an important tool to confirm the clinical diagnostic. Molecular biology and biotechnological methods have been used to assist in the diagnosis of this disease, each one with its advantages and drawbacks. Enzyme-linked immunosorbent assay (ELISA) is the used method for leprosy diagnosis, and it allows the detection of infection-related antigens. Alternatively, due to their versatility to perform the same functions as the protein and non-protein natural antigens, mimetic peptides are considered an important tool. On the other hand, lateral flow assay (LFA) and optical and electrochemical biosensors are rapid and portable methods, capable of performing diagnosis in the field without sample preparation.This chapter presents such techniques, their uses in the diagnosis and detection of M. leprae, as well as the potential for the development of new techniques and strategies that can help to control and understand mycobacteriosis.

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qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients

Abstract: Background

Cited by 17 publications

References 19 publications

In silico analysis of V48A dihydropteroate synthase mutation to dapsone on Mycobacterium leprae from Papua

In silico analysis of V48A dihydropteroate synthase mutation to dapsone on Mycobacterium leprae from Papua

Nested PCR and the TaqMan SNP Genotyping Assay enhanced the sensitivity of drug resistance testing of Mycobacterium leprae using clinical specimens of leprosy patients

Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

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