qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Azevedo, Michelle de Campos Soriani; Ramuno, Natália Mortari; Fachin, Luciana Raquel Vincenzi; Tassa, Mônica; Rosa, Patrícia Sammarco; Belone, Andréa de Faria Fernandes; Diório, Suzana Madeira; Soares, Cléverson Teixeira; Garlet, Gustavo; Trombone, Ana Paula

doi:10.1016/j.bjid.2016.09.017

Cited by 38 publications

(42 citation statements)

References 15 publications

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“…The blood samples showed, overall, 90.2% positivity in the PCR and qPCR reactions, using the new primer pair. These results were improved in comparison to those obtained from biopsies analysed with the LP1–LP2 primer pair, which showed 84·9% of positivity (Azevedo et al ). Moreover, for diagnostic tests, biopsy specimens are poorly indicated because it is an invasive technique and is not widely used (Scollard et al ; Johnson et al ).…”

Section: Discussionmentioning

confidence: 76%

“…Many studies have shown that PCR is an important methodology for leprosy’s diagnosis and cure control (Azevedo et al ; Soto et al ). Some authors have demonstrated that primers for the RLEP repeating sequence are successfully used to amplify M. leprae ’s DNA from leprosy patients at different stages of the disease, demonstrating that they had a higher positivity than primers from the 16SrRNA ribosomal region (León et al ).…”

Section: Discussionmentioning

confidence: 99%

“…However, despite its high specificity, sensitivity is variable. To increase M. leprae’s detection, authors have searched for new methods, such as molecular markers based on the polymerase chain reaction (PCR) technique, which is faster, more sensitive and specific, and could represent an alternative for the diagnosis and identification of this pathogen (Azevedo et al ). Distinct M. leprae ’s genome sequences, such as RLEP, 16S rRNA, 16STR and 18 kDa regions have been evaluated and studies demonstrated that primers for the RLEP repeating sequence are successfully used to amplify M. leprae ’s DNA from patients at different stages of the disease, demonstrating more positive results (Donoghue et al ).…”

Section: Introductionmentioning

confidence: 99%

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Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

Machado¹,

Lyon

Rocha-Silva³

et al. 2020

J Appl Microbiol

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Aims Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae’s DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. Methods and Results The specific target region, RLEP, of M. leprae’s genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. Conclusions In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. Significance and Impact of the Study The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.

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Section: Discussionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

Machado¹,

Lyon

Rocha-Silva³

et al. 2020

J Appl Microbiol

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“…Currently, several quantitative PCR tests have been optimized for detection of M . leprae but the cost and the absence of a standardized protocol is a limitation to its implementation at lower level health institutions in resource-limited countries [ 44 , 46 , 47 ].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Auramine O staining and conventional PCR for leprosy diagnosis: A comparative cross-sectional study from Ethiopia

et al. 2018

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BackgroundDiagnosis of leprosy mainly relies on clinical examination due to the inconsistent sensitivity and poor reproducibility of the current laboratory tests. Utilisation of alternative methods to the standard Ziehl Neelsen (ZN), Fite-Faraco (FF) and Haematoxylin and Eosin (H&E) staining procedures may eventually improve leprosy diagnosis.Methodology/Principal findingsIn this comparative study, the performance of the fluorescent Auramine O (AO) staining and polymerase chain reaction (PCR) was assessed with different skin samples using a combination of ZN, FF and H&E staining as the gold standard. AO, ZN, FF, H&E and PCR tests were performed on slit skin smears (SSS) and/or punch biopsies collected from 141 clinically confirmed leprosy cases and 28 non-leprosy skin samples. DNA was extracted from punch biopsies using two different methods with or without mechanical lysis.Sensitivities were 87.6%, 59.3% and 77% for H&E, ZN and FF, respectively, whereas it reached 65.5% and 77.9% for AO in SSS and tissue sections and 91.1% for PCR in tissue samples. Morover, samples with low bacillary index, sensitivity of AO staining (61.8%) was similar to FF (60%, p>0.05) and lower than PCR (86.6%, p<0.05). Sensitivity of PCR also increased (96.8%, p<0.05) when mechanical lysis was used during DNA extraction compared to enzymatic treatment alone (84.6%).Conclusions/SignificanceOur results showed that for diagnostic purposes, analysis of skin section is more sensitive than SSS, especially for samples with low bacillary load. AO staining on SSS and tissue sections was not significantly better than other routine diagnostic tests but considerably more user friendly. The sensitivity of PCR was higher than current standard methods and increased when combined with more efficient DNA extraction using mechanical and chemical lysis. Therefore, we recommend AO staining for the diagnosis of leprosy in lower health facilities such as health centres and district hospitals and PCR diagnosis at referral level and research centres.

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“…At each concentration, three replicates were tested in a single run. The following data were determined as estimators of the amplification efficiency: already been demonstrated as having higher sensitivities than the sensitivity observed for bacilloscopy of skin biopsies and slit-skin smears [8,[10][11][12].…”

Section: Amplification Efficiency and Limit Of Detection (Lod) Of Qpcrmentioning

confidence: 99%

Evaluation of 16S rRNA qPCR for detection ofMycobacterium lepraeDNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases

Marques

Frota

Quetz

et al. 2017

Pathogens and Global Health

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Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (T = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 10-8.02 × 10, and in SB samples from MB patients were 1.87 × 10-1.50 × 10. Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

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qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Cited by 38 publications

References 15 publications

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy

Evaluation of Auramine O staining and conventional PCR for leprosy diagnosis: A comparative cross-sectional study from Ethiopia

Evaluation of 16S rRNA qPCR for detection ofMycobacterium lepraeDNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases

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