Dataset Information

Comparison of Three PCR-based Methods for Simplicity and Cost Effectiveness Identification of Cutaneous Leishmaniasis Due to Leishmania tropica.

ABSTRACT: BACKGROUND:To compare three molecular methods, PCR-RFLP for internal transcribed spacer, PCR sequencing and high resolution melting analysis shown reliable sensitivity and specificity for detecting Leishmania tropica as a model for cutaneous leishmaniasis (CL) as the perspective overview for scientific and economic approaches. METHODS:This study was carried out between 2015 and 2016 in Leishmaniasis Research Center in Kerman University of Medical Sciences, Kerman, Iran. The positives smears (n=50) were obtained from patients referred from the health clinics in a major anthroponotic CL (ACL) focus, southeastern Iran. Only smear preparations with the same grade were selected according to the method described by the WHO for future PCR assays. RESULTS:All three molecular methods had capability to identify positive samples at species level with the same specificity and sensitivity. However, these techniques were different in simplicity, consuming time, and cost effectiveness. Although additional enzymatic process in PCR-RFLP provided good resolution to find Leishmania species but this would cause time and cost increases. CONCLUSION:HRM (high resolution melting) is a relatively new technique that allows direct characterization of PCR amplicons in a closed system with more simplicity, cost effectiveness and time-consuming compared with other PCR-based assays for epidemiological or clinical identification purposes.

SUBMITTER: Mohammadi MA

PROVIDER: S-EPMC5527031 | biostudies-literature | 2017 Apr-Jun

REPOSITORIES: biostudies-literature

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Comparison of Three PCR-based Methods for Simplicity and Cost Effectiveness Identification of Cutaneous Leishmaniasis Due to <i>Leishmania tropica</i>.

Mohammadi Mohammad Ali MA Bamorovat Mehdi M Fasihi Harandi Majid M Karimi Tayyebeh T Sharifi Iraj I Aflatoonian Mohammad Reza MR

Iranian journal of parasitology 20170401 2

<h4>Background</h4>To compare three molecular methods, PCR-RFLP for internal transcribed spacer, PCR sequencing and high resolution melting analysis shown reliable sensitivity and specificity for detecting <i>Leishmania tropica</i> as a model for cutaneous leishmaniasis (CL) as the perspective overview for scientific and economic approaches.<h4>Methods</h4>This study was carried out between 2015 and 2016 in Leishmaniasis Research Center in Kerman University of Medical Sciences, Kerman, Iran. The ...[more]

PMID: 28761481

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Project description:BackgroundRecent circumstantial evidence suggests that an increasing number of Iranian patients with cutaneous leishmaniasis are unresponsive to meglumine antimoniate (Glucantime), the first line of treatment in Iran. This study was designed to determine whether the clinical responses (healing, or non-healing) were correlated with the susceptibility of Leishmania parasites to Glucantime.Methods and findingsIn vitro susceptibility testing was first performed on 185 isolated parasites in the intracellular mouse peritoneal macrophage model. A strong correlation between the clinical outcome and the in vitro effective concentration 50% (EC50) values was observed. Parasites derived from patients with non-healing lesions had EC50 values at least 4-fold higher than parasites derived from lesions of healing patients. A selection of these strains was typed at the molecular level by pulsed-field gels and by sequencing the pteridine reductase 1 (PTR1) gene. These techniques indicated that 28 out of 31 selected strains were Leishmania tropica and that three were Leishmania major. The L. major isolates were part of a distinct pulsed-field group, and the L. tropica isolates could be classified in three related additional pulsed-field groups. For each pulsed-field karyotype, we selected sensitive and resistant parasites in which we transfected the firefly luciferase marker to assess further the in vitro susceptibility of field isolates in the monocyte cell line THP1. These determinations confirmed unequivocally that patients with non-healing lesions were infected with L. tropica parasites resistant to Glucantime. Additional characterization of the resistant isolates showed that resistance is stable and can be reversed by buthionine sulfoximine, an inhibitor of glutathione biosynthesis.ConclusionsTo the authors' knowledge, this is the first report of proven resistant parasites contributing to treatment failure for cutaneous leishmaniasis and shows that primary Glucantime-resistant L. tropica field isolates are now frequent in Iran.

Project description:BackgroundDrug resistance is a common phenomenon frequently observed in countries where leishmaniasis is endemic. Due to the production of the pteridine reductase enzyme (PTR1), drugs lose their efficacy, and consequently, the patient becomes unresponsive to treatment. This study aimed to compare the in vitro effect of meglumine antimoniate (MA) on non- healing Leishmania tropica isolates and on MA transfected non-healing one to PTR1.MethodsTwo non-healing and one healing isolates of L. tropica were collected from patients who received two courses or one cycle of intralesional MA along with biweekly liquid nitrogen cryotherapy or systemic treatment alone, respectively. After confirmation of L. tropica isolates by polymerase chain reaction (PCR), the recombinant plasmid pcDNA-rPTR (antisense) was transfected via electroporation and cultured on M199. Isolates in form of promastigotes were treated with different concentrations of MA and read using an enzyme-linked immunosorbent assay (ELISA) reader and the half inhibitory concentration (IC50 ) value was calculated. The amastigotes were grown in mouse macrophages and were similarly treated with various concentrations of MA. The culture glass slides were stained, and the mean number of intramacrophage amastigotes and infected macrophages were assessed in triplicate for both stages.ResultsAll three transfected isolates displayed a reduction in optical density compared with the promastigotes in respective isolates, although there was no significant difference between non-healing and healing isolates. In contrast, in the clinical form (amastigotes), there was a significant difference between non-healing and healing isolates (p < 0.05).ConclusionThe results indicated that the PTR1 gene reduced the efficacy of the drug, and its inhibition by antisense and could improve the treatment of non-healing cases. These findings have future implications in the prophylactic and therapeutic modality of non- healing Leishmania isolates to drug.

Dataset Information

Comparison of Three PCR-based Methods for Simplicity and Cost Effectiveness Identification of Cutaneous Leishmaniasis Due to Leishmania tropica.

Publications

Comparison of Three PCR-based Methods for Simplicity and Cost Effectiveness Identification of Cutaneous Leishmaniasis Due to <i>Leishmania tropica</i>.

Similar Datasets

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