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

ABSTRACT: 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±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C 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.

SUBMITTER: Khademvatan S

PROVIDER: S-EPMC4089809 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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

Khademvatan S S Neisi N N Maraghi S S Saki J J

Annals of tropical medicine and parasitology 20111201 8

<h4>Background</h4>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.<h4>Materials and methods</h4>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 fluor ...[more]

PMID: 22325815

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Project description:BackgroundQuery ID="Q1" Text="Graphical abstract: As per journal requirements, graphical abstract is necessary. Kindly check and provide the same."The magnitude of the health problems caused by leishmaniasis has been a major driving factor behind the development and implementation of leishmaniasis control programs by the national authorities in Iran, with a priority for health and environmental management. Such programs are not achievable unless all of the factors leading to the infection, including the parasite's life-cycle, vectors and reservoirs, are recognized. So far in Iran, humans and rodents have been considered the principal reservoirs of Leishmania tropica and Leishmania major, respectively, both associated with cutaneous leishmaniasis (CL), with domestic dogs considered to be the main reservoir for Leishmania infantum, associated with visceral leishmaniasis (VL). The role of other mammals in maintaining the Leishmania parasite has remained unclear. This study aimed to investigate Leishmania infection among livestock in endemic areas of VL and CL in Fars province, southern Iran, using serological and molecular methods.MethodsBlood samples from 181 clinically healthy livestock, including 49 sheep, 114 goats, 16 cattle and two donkeys, were screened to detect Leishmania DNA and anti-Leishmania antibodies using qPCR (quantitative PCR) and the direct agglutination test (DAT), respectively. Four qPCR-positive samples were amplified using the internal transcribed spacer one (ITS1) primers in conventional PCR and sent for directional sequencing.ResultsOf the 181 livestock tested, 51 (28.2%) were infected with Leishmania, using serological and molecular methods. Anti-Leishmania antibodies were detected in 70 (38.7%) (95% confidence interval [CI]: 31.5-46.2) and Leishmania DNA in 93 (51.4%) (95% CI: 43.9-58.9) livestock. The identified Leishmania spp. were L. infantum and L. major.ConclusionsThe findings of the present study show a relatively high prevalence of Leishmania infection among livestock in endemic areas of the disease, in Fars province, southern Iran. Given the large population of this group of animals and the fact that they live in the vicinity of the main reservoirs of the disease and vectors, it seems that sand flies regularly bite these animals. Further studies are needed to determine the role of livestock in the parasite's life-cycle and the epidemiology of Leishmania infection.

Project description:BackgroundKRAS mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect KRAS mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for KRAS mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes.MethodsWe developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in KRAS codons 12 and 13 using the Roche LightCycler® instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (Tc) of 81°C increased the sensitivity of the assay >10-fold for the majority of KRAS mutations.ResultsWe compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant KRAS mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background.ConclusionsWe have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for KRAS mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.

Dataset Information

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

Publications

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

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