sybr-green-i and Leishmaniasis--Cutaneous

Cutaneous leishmaniasis (CL) is an important public health problem, most frequently seen in Şanlıurfa in Turkey. It is important to determine the species in regions where infection occurs with different Leishmania species, as in our province. In this study, it was aimed to genotype 136 samples with suspected Leishmania from Şanlıurfa using the Sybr Green-based ITS-1 real time polymerase chain reaction (Rt-PCR) method and then to compare them with ITS-1 PCR RFLP and direct microscopy methods. Wound fluid samples from patient lesions suspected of leishmaniasis were mounted on a slide, fixed, and stained with Giemsa dye. The preparations were examined under the microscope and evaluated for the presence of amastigote. After the extraction of DNA from Giemsa stained preparations by using the QIAmp DNA Mini Kit (Qiagen, Germany), the samples were studied with the Sybr Green based ITS-1 Rt-PCR method using LITSR and L5.8S primers. As a result of the PCR study, melting curve analysis was determined and the melting curves were compared with the reference strains. Then, PCR was performed in 136 samples for ITS1 region amplification using primers LITSR and L5.8S. PCR products were digested with Hae III restriction enzyme and RFLP process was performed. The products were run on metaphor agarose gel than the gels were stained with ethidium bromide for 15 min and visualized in a UV transilluminator In our study, the results of Sybr Green-based ITS-1 Rt-PCR, ITS-1 PCR-RFLP and direct microscopy methods were compared. The highest positivity rate was determined as 97% (136/132) in ITS-1 Rt-PCR method. With ITS-1 PCR-RFLP method 95.5% (136/130) positivity and with direct microscopy 94.1% (136/128) positivity were obtained, respectively. Of 132 samples, which were studied with the Sybr Green-based ITS-1 Rt-PCR method and found as positive, 121 were genotyped as L.tropica and 11 were genotyped as L.major by melting curve analysis. It was determined that, of 130 samples studied with ITS-1 PCR RFLP method 119 (91.5%) were detected as L.tropica and 11 (8.5%) were detected as L.major. The ITS-1 Rt-PCR method we used in our study was the method that detected the most positivity rate. With this method, Leishmania specimens were typed as L.tropica and L.major. It is thought that this method may be useful for the detection of the presence of Leishmania parasite and in the rapid identification of Leishmania species, as it does not require extra processes such as cutting and staining

Topics: Benzothiazoles; Diamines; DNA Primers; DNA, Protozoan; Humans; Leishmania; Leishmaniasis, Cutaneous; Quinolines; Real-Time Polymerase Chain Reaction

Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania) infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1), whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2). The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania) and Leishmania (Viannia) using the qPCR2 assay followed by melting or High Resolution Melt (HRM) analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania) and Leishmania (Viannia) subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L.) infantum WHO international reference strain (MHOM/TN/80/IPT1), highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical samples, must be taken into account in quantitative PCR-based applications; however, it might also be used to differentiate between Leishmania subgenera.

Topics: Animals; Benzothiazoles; Diagnosis, Differential; Diamines; DNA Copy Number Variations; DNA Primers; DNA, Kinetoplast; Dog Diseases; Dogs; Fluorescent Dyes; Leishmania braziliensis; Leishmania infantum; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Nucleic Acid Denaturation; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity

Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk.. A SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95 °C ± 0.01 and 77.36 °C ± 0.02 for Leishmania and Viannia, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together L. (V.) braziliensis and L. (V.) shawii with a bootstrap value of 100%; while for L. (L.) infantum and L. (L.) amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus Leishmania (average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities.. For all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.

Topics: Adolescent; Animals; Base Sequence; Benzothiazoles; Brazil; Child; Child, Preschool; Conserved Sequence; Diamines; DNA, Kinetoplast; Female; Fluorescent Dyes; Humans; Insect Vectors; Leishmania; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Male; Molecular Sequence Data; Organic Chemicals; Phylogeny; Psychodidae; Quinolines; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Sequence Alignment; Sequence Analysis, DNA