Advantages and limits of real-time PCR assay and PCR-restriction fragment length polymorphism for the identification of cutaneous Leishmania species in Tunisia

Abda, I. Ben; Monbrison, Frédérique de; Bousslimi, Nadia; Karim, Abdul; Bouratbine, A.; Picot, Stéphane

doi:10.1016/j.trstmh.2010.09.003

Cited by 34 publications

(23 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The PCR and real-time PCR methods have recently been reported as having a high sensitivity in the diagnosis of CCL, and the sensitivity of real-time PCR has been suggested to be more than that of the PCR. 25,26 When PCR and real-time PCR methods were compared in diagnosis of CCL, the results of our study confirmed the results of the previous studies.…”

Section: Laboratory Diagnosis In Chronic Cutaneous Leishmaniasissupporting

confidence: 88%

Comparison of Clinical Samples and Methods in Chronic Cutaneous Leishmaniasis

Eroglu¹,

Uzun²,

Koltas³

2014

The American Society of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

Abstract. This study aimed at finding out the most effective clinical samples and methods in chronic cutaneous leishmaniasis (CCL). Smear, aspiration fluid, and filter paper samples were taken from 104 skin lesions of suspected cases with CCL, and they were compared using microscopic examination, culture, and molecular methods. We characterized four different forms of CCL and identified the causative agents in CCL forms using high-resolution melting curve real-time polymerase chain reaction assay. We observed that smear was detected to be the most sensitive (63.5%) among clinical samples, and real-time polymerase chain reaction method was the most sensitive (96.8%) among the methods used in diagnosis of CCL. We identified 68.8% Leishmania tropica and 31.2% L. infantum in papular lesions, 69.2% L. infantum and 30.8% L. tropica in nodular lesions, 57.9% L. tropica and 42.1% L. major in ulcerating plaque lesions, and 55.5% L. tropica and 44.5% L. major in noduloulcerative lesions in CCL patients.

show abstract

Section: Laboratory Diagnosis In Chronic Cutaneous Leishmaniasissupporting

confidence: 88%

Comparison of Clinical Samples and Methods in Chronic Cutaneous Leishmaniasis

Eroglu¹,

Uzun²,

Koltas³

2014

The American Society of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

show abstract

“…Although SYBR Green qPCR detected 2 ATL cases among 15 cases in which Leishmania was not found using the CRS, conventional PCR and qPCR performed similarly ( Table 3). The main advantage of qPCR is its greater automation, which results in time savings (25)(26)(27).…”

Section: Discussionmentioning

confidence: 99%

Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

et al. 2017

View full text Add to dashboard Cite

The precise diagnosis of American tegumentary leishmaniasis (ATL) is an essential task due to the disease's associated morbidity. A noninvasive, extremely sensitive, and highly specific exam is critical, particularly for mucosal leishmaniasis (ML), in which a low parasite quantity is expected. We aimed to compare the diagnostic accuracy of swab and biopsy sample analysis using SYBR Green-and TaqManbased real-time PCR (qPCR) assays with that of a composite reference standard consisting of the Montenegro skin test, serology, histopathology, smears, culture, and conventional PCR. In total, 55 patients with ATL (ML, 18 patients; cutaneous leishmaniasis [CL], 37 patients) and 36 patients without ATL were studied. qPCR analysis of swabs was more accurate when using SYBR Green (87.88%; 95% confidence interval [CI], 77.86 to 93.73 patients) than when using TaqMan (78.79%; 95% CI, 67.49 to 86.92%) (P ϭ 0.031). SYBR Green (84.72%; 95% CI, 74.68 to 91.25%) was also more accurate than TaqMan (73.61%; 95% CI, 62.42 to 82.41%) for biopsy samples (P ϭ 0.008). All qPCR methods were 100% specific. Swabs and biopsy specimens had similar sensitivity when using the same chemistry (P ϭ 0.125 for SYBR Green and P ϭ 0.625 for TaqMan). Moreover, qPCR achieved better performance than most existing techniques used for the diagnosis of ATL and also detected the Leishmania parasite in a greater proportion of patients than the associated histopathology, smear, culture, and conventional PCR techniques did. Swabs therefore represent a useful diagnostic tool because they not only are noninvasive but also can achieve an accuracy similar to that of biopsy samples. The high accuracy of SYBR Green-based qPCR may also reduce the requirement for associated parasitological tests for ATL diagnosis.

show abstract

“…The relatively new method real-time PCR can process a sample in less than an hour, has been reported to rapidly diagnosis and differentiate single nucleotide mutations within a target DNA sequence (Wortmann et al, 2005;Castilho et al, 2008;TalmiFrank et al, 2010;Ben Abda et al, 2011). This assay has the potential to provide the treating clinician with a rapid identification of the infecting Leishmania species, which subsequently offers the potential for targeted treatment strategies (Wortmann et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Diagnosis and identification ofLeishmaniaspp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran

Khademvatan

Neisi

Maraghi

et al. 2011

Annals of Tropical Medicine & Parasitology

View full text Add to dashboard Cite

Background:The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran.Materials and methods: Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis.Results: One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T m ) were 88.3¡0.2uC for L. tropica (MHOM/IR/02/Mash10), 86.5¡0.2uC for L. major (MHOM/IR/75/ER) and 89.4¡0.3uC for L. infantum (MCAN/IR/97/LON 49), respectively.Conclusion: This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.

show abstract

Advantages and limits of real-time PCR assay and PCR-restriction fragment length polymorphism for the identification of cutaneous Leishmania species in Tunisia

Cited by 34 publications

References 33 publications

Comparison of Clinical Samples and Methods in Chronic Cutaneous Leishmaniasis

Comparison of Clinical Samples and Methods in Chronic Cutaneous Leishmaniasis

Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

Diagnosis and identification ofLeishmaniaspp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran

Contact Info

Product

Resources

About