Molecular-Genotyping Detection of Entamoeba histolytica in Diarrheic Patients.

Amebiasis is caused by Entamoeba histolytica, a protozoan that is found worldwide. The degree of pathogenesis of clinical isolates varies greatly. This study was aimed to molecular identification of E. histolytica in children using the nested polymerase chain reaction (nPCR), and then, a genotyping of positive E. histolytica isolates using the quantitative PCR (qPCR) assay through targeting serine-rich E. histolytica protein (SREHP) gene. For this purpose, a total of 50 bloody diarrheic stool samples were collected from the children attended to the Al-Zahraa' Teaching Hospital and Alkut Hospital for Gynecology, Obstetric and Pediatrics (Alkut, Wasit, Iraq) were subjected to the present study from September to December 2021. Firstly, the extracted DNAs that amplified using specific primers through targeting 18S rRNA gene and tested using nPCR assay were revealed an overall 48% (24/50) positive samples for E. histolytica. For genotyping, our results were detected an existence of four different genotypes (I, II, III and IV) with a significant prevalence of Genotype-II (54.17%) when compared to Genotype-I (20.83%), Genotype-III (12.5%) and Genotype- IV (12.5%). In addition, results of melting temperature of targeted genotypes were 84ºC, 83 - 83.5ºC, 82.5ºC and 81ºC for Genotype-I, II, III and IV, respectively. In conclusion, molecular amplification of 18S rRNA gene was revealed the large prevalence of E. histolytica among bloody diarrheic children of study areas; while, amplification of SREHP gene was reflected the widespread phenotypic variation of the Genotype-II suggesting the high ability of this genotype to spread infection in children. In different endemic areas as Iraq, the utilization of high-resolution genotyping methods showed the extremely polymorphic genetic structure of this parasite.

Evaluation of curcumin and CM11 peptide alone and in combination against amastigote form of Iranian strain of L. major (MRHO/IR75/ER) in vitro.

Curcumin (diferuloylmethane) is the main phytochemical of Curcuma longa Linn, an extract of the rhizome turmeric. For thousands of years, turmeric among other natural products has been used as a dietary spice and as a medicinal plant in Asian countries. The present study reports the leishmanicidal activity of curcumin in different concentrations (10 µM, 20 µM, 40 µM). It is also showing the effect of CM11 peptide (8 µM) alone and in combination with curcumin (10 and 20 µM) as a leishmanicidal drug. The experiments were performed with the amastigote form of Leishmania major (MRHO/IR/75/ER) in vitro and the leishmanicidal activity was analyzed after 12 and 24 h of incubation by Giemsa and DAPI staining. Further investigation was done by using semi-quantitative PCR with new designed common primer pair derived from an 18S rRNA gene belonging to the L. major and mouse, which amplified the above-mentioned gene segments simultaneously with different PCR product size. Our findings showed that curcumin had leishmanicidal activity in a dose and time-dependent manner and its lowest effective dose was at concentrations of 40 µM afetr12 h and 10 µM after 24 h. The IC50 value of curcumin against amastigote forms of L. major was 21.12 µM and 11.77 µM after 12 and 24 h, respectively. Treatment of amastigote form with CM11 (8 µM) alone and in combination with curcumin (10 µM and 20 µM) showed less leishmanicidal activity. Interestingly, CM11 in combination with curcumin (10 µM and 20 µM) had even less leishmanicidal effect compared to curcumin alone in the same concentrations (10 µM and 20 µM). The semi-quantitative PCR analysis confirmed the data achieved by Giemsa and DAPI staining and showed that curcumin reduced the PCR product derived from amastigote form in concentration and time-dependent manner compared to the genome of the host cells.

Determination of the Effective Dose of Curcumin alone and in Combination with Antimicrobial Peptide CM11 on Promastigote Forms of Iranian Strain of L. major (MRHO / IR / 75 / ER).

Zoonotic cutaneous leishmaniasis t caused by Leishmania major is spread in focal areas of more than 90 countries in the tropics, subtropics, and southern Europe. In the absence of any effective vaccine, the only means to treat and control leishmaniasis is conventional medication. Glucantime is the first choice of anti-leishmanialdrug, has serious side effects like high toxicity, exorbitant cost, problems with the administration and development of resistance. Curcumin is the active component from the rhizome of herb Curcuma longa, possessing many pharmacological and biological activities with antiprotozoal and anti-proliferative effects which make it a good alternative to existing therapy. Antimicrobial peptides like CM11, a small peptide consisting of 11 amino acids, are also novel potential drugs against at least wide spectrum of microbial organisms. The aim of this study was to evaluate the effect of curcumin alone and in combination with CM11 on promastigote form of L. major (MRHO / IR / 75 / ER) for 12h and 24h in vitro. The results of Giemsa staining showed that the morphology of the flagellum and cell shape increased changed with increasing concentration of curcumin (5 µM, 10 μM, 20 μM, 40 μM and 80 μM). MTT and Trypan blue results demonstrated that the promastigotes were susceptible against curcumin in dose and time dependent manner, while CM11 alone at concentration of 8 µM as well as in combination with 10 and 20 µM curcumin had no significant effect on promastigotes. Our results revealed that curcumin can provide a new curative candidate against cutaneous leishmaniasis.