DNA extraction and amplification of<i>Leishmania</i>from archived, Giemsa-stained slides, for the diagnosis of cutaneous leishmaniasis by PCR

Motazedian, H; Karamian, Mehdi; Noyes, Harry; Ardehali, S.

doi:10.1179/000349802125000484

Cited by 82 publications

(68 citation statements)

References 6 publications

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“…The use of archived Giemsa-stained slides for molecular diagnosis and differentiation of causative agent of CL has been described in several studies (Motazedian et al, 2002;AlJawabreh et al, 2006;Kazemi-Rad et al, 2008). Motazedin et al indicate that the PCR-based checking of Giemsa-stained smears appears to be reasonably sensitive and specific in revealing the presence of Leishmania parasites (or, at least undegraded leishmanial DNA) in such chronic lesions (Motazedian et al, 2002).…”

Section: Discussionmentioning

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

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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.

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Section: Discussionmentioning

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

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“…Total DNA was extracted from blood buffy coat as described by Motazedian et al (2002). Briefly, 200 ll of buffy coat was homogenized with 200 ll lyses buffer [50 mM TrisHCl (pH = 7.6), 1 mM EDTA and 1 % Tween 20] and 10 ll of proteinase K solution (containing 19 mg of the enzyme/ ml), in a 1.5 ml micro centrifuge tube.…”

Section: Sample Collection and Testingmentioning

confidence: 99%

Emergence of a new focus of visceral leishmaniasis due to Leishmania infantum in Golestan Province, north-eastern of Iran

et al. 2013

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Over the last decade, a few cases of visceral leishmaniasis (VL) have been reported in some districts of the province of Golestan, in north-eastern Iran. The aim of the present study was to investigate the prevalence of Leishmania infantum infection among humans and domestic dogs by using direct agglutination test (DAT) and PCR assays in the eastern zone of the province. Between 2011 and 2012, blood samples were randomly collected from 450 humans and 50 domestic dogs, in the eastern zone of Golestan Province including 7 villages from Maravetappeh district where new cases of human VL had been recorded there. Each of these samples was tested for antiLeishmania antibodies, in DAT, and for L. infantum kinetoplast DNA on whole blood, in PCR-based assays. A total of 450 human samples, 6 (1.33 %) were found seropositive and 13 (2.8 %) was found PCR-positive. Of the 50 dog samples, 16 (32 %) were found seropositive and 15 (30 %) were PCR-positive. All PCR-positive dogs were found seropositive except one as well as 6 (46.2 %) PCR-positive humans were also found seropositive. Moreover, the species of L. infantum was detected in all PCR-positive samples. The high prevalence of VL in the study areas offer it has emerged as an endemic focus in the province. Further investigations on the vectors, reservoirs and human population are recommended.

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“…The entire smear was then scraped off the slide, with a sterile scalpel, so that the total DNA in the smear could be extracted by digestion, in a 1.5-ml microcentrifuge tube, with 200 ml lysis buffer [50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 1% (v/v) Tween 20] containing 8.5 ml of a proteinase-K solution that had 19 mg enzyme/ml (Yokota et al, 1995;Motazedian et al, 2002). The tube was incubated overnight, at 37uC, before 200 ml phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.)…”

Section: Dna Extractionmentioning

confidence: 99%

“…The resultant supernatant solution was transferred to another tube and mixed with 400 ml absolute ethanol. The DNA that precipitated was centrifuged down (at 60006g for 20 min), dried, dissolved in 50 ml ultrapure water [produced in a PurelabH UHQ system (Siemens Water Technologies, Warrendale, PA)], and stored at 4uC (Motazedian et al, 2002) before use in the PCR-based assay.…”

Section: Dna Extractionmentioning

confidence: 99%

The PCR-based detection and identification of the parasites causing human cutaneous leishmaniasis in the Iranian city of Ahvaz

Ghasemian

Maraghi

Samarbafzadeh

et al. 2011

Annals of Tropical Medicine & Parasitology

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In Iran, Leishmania major or L. tropica cause almost all of the human cutaneous leishmaniasis (CL). Unfortunately, the detection methods frequently used for CL (the microscopical examination of direct smears or the culture of biopsies) are not very sensitive and the Leishmania species causing each case of CL in Iran is usually only tentatively identified from extrinsic factors, such as the case's clinical manifestations and region of residence. Recently, however, a nested PCR that targets the parasites' kinetoplast DNA has been used in the city of Ahvaz (the capital of the province of Khouzestan, in south-western Iran) to confirm the microscopical diagnosis of CL and to identify the causative parasites, to species level. Smears from the lesions on 100 suspected cases of CL were fixed, stained with Wright's eosin-methylene blue, and checked for amastigotes under a light microscope. Scrapings from the same smears were then tested for leishmanial DNA, using a nested PCR that allows the DNA from L. tropica to be identified and distinguished from that of L. major. The 100 smears investigated were all found amastigote-positive by microscopy and PCR-positive for either L. major DNA (97 smears) or L. tropica DNA (three smears). The predominant species causing CL in Ahvaz is therefore L. major.

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DNA extraction and amplification ofLeishmaniafrom archived, Giemsa-stained slides, for the diagnosis of cutaneous leishmaniasis by PCR

Cited by 82 publications

References 6 publications

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

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

Emergence of a new focus of visceral leishmaniasis due to Leishmania infantum in Golestan Province, north-eastern of Iran

The PCR-based detection and identification of the parasites causing human cutaneous leishmaniasis in the Iranian city of Ahvaz

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