An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis

Cruz, Israel; Millet, Aurélie; Carrillo, Eugenia; Chenik, Mehdi; Salotra, Poonam; Verma, Sandeep; Veland, Nicolás; Jara, Marlene; Adaui, Vanessa; Castrillón, Carlos; Arévalo, Jorge; Moreno, Javier; Cañavate, Carmen

doi:10.1016/j.exppara.2013.03.026

Cited by 62 publications

(58 citation statements)

References 49 publications

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“…They were applied for species discrimination by RFLP analysis with the enzyme HaeIII by Schönian et al (112), who evaluated the technique in clinical samples. It allowed separation of species from the Leishmania (Leishmania) subgenus but did not discriminate Leishmania (Viannia) species (77,113). The authors evaluated their method using only one type strain of each investigated species (Table 1), thereby overlooking possible intraspecies variation that could lead to erroneous results.…”

Section: Rdna Arraymentioning

confidence: 99%

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Species Typing in Dermal Leishmaniasis

2015

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SUMMARY Leishmania is an infectious protozoan parasite related to African and American trypanosomes. All Leishmania species that are pathogenic to humans can cause dermal disease. When one is confronted with cutaneous leishmaniasis, identification of the causative species is relevant in both clinical and epidemiological studies, case management, and control. This review gives an overview of the currently existing and most used assays for species discrimination, with a critical appraisal of the limitations of each technique. The consensus taxonomy for the genus is outlined, including debatable species designations. Finally, a numerical literature analysis is presented that describes which methods are most used in various countries and regions in the world, and for which purposes.

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Section: Rdna Arraymentioning

confidence: 99%

“…The first stage usually targets detection of the Leishmania genus rather than a specific species (20,(74)(75)(76)(77). Assays focusing on this level are not covered here, except when species typing relies on downstream analysis of PCR amplification products generated in the detection process.…”

Section: Levels Of Typing and Focus Of This Reviewmentioning

confidence: 99%

Species Typing in Dermal Leishmaniasis

2015

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“…Primer pair choices were based on previously described sequences that amplify fragments of the Leishmania (V.) braziliensis minicircle kDNA (5,(32)(33)(34)(35)(36). We evaluated primer and probe specificity for Leishmania (V.) braziliensis using NCBI BLAST.…”

Section: Standardization Tests (Index Tests)mentioning

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

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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.

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“…The advantages of real-time quantitative PCR (qPCR) approaches in comparison with conventional PCR assays include the potential for increased sensitivity and specificity and for a decreased risk of contamination of the laboratory environment (14,(18)(19)(20)(21)(22)(23)(24)(25)(26). The efficiency of real-time platforms for Leishmania species identification has been discussed (24,(27)(28)(29)(30).…”

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confidence: 99%

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

et al. 2017

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Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia. Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (T m ) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the T m values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania

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An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis

Cited by 62 publications

References 49 publications

Species Typing in Dermal Leishmaniasis

Species Typing in Dermal 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

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

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