Differential Expression and Function of SVIP in Breast Cancer Cell Lines and In Silico Analysis of Its Expression and Prognostic Potential in Human Breast Cancer.

The heterogeneity of cancer strongly suggests the need to explore additional pathways to target. As cancer cells have increased proteotoxic stress, targeting proteotoxic stress-related pathways such as endoplasmic reticulum stress is attracting attention as a new anticancer treatment. One of the downstream responses to endoplasmic reticulum stress is endoplasmic reticulum-associated degradation (ERAD), a major degradation pathway that facilitates proteasome-dependent degradation of unfolded or misfolded proteins. Recently, SVIP (small VCP/97-interacting protein), an endogenous ERAD inhibitor, has been implicated in cancer progression, especially in glioma, prostate, and head and neck cancers. Here, the data of several RNA-sequencing (RNA-seq) and gene array studies were combined to evaluate the SVIP gene expression analysis on a variety of cancers, with a particular focus on breast cancer. The mRNA level of SVIP was found to be significantly higher in primary breast tumors and correlated well with its promoter methylation status and genetic alterations. Strikingly, the SVIP protein level was found to be low despite increased mRNA levels in breast tumors compared to normal tissues. On the other hand, the immunoblotting analysis showed that the expression of SVIP protein was significantly higher in breast cancer cell lines compared to non-tumorigenic epithelial cell lines, while most of the key proteins of gp78-mediated ERAD did not exhibit such an expression pattern, except for Hrd1. Silencing of SVIP enhanced the proliferation of p53 wt MCF-7 and ZR-75-1 cells but not p53 mutant T47D and SK-BR-3 cells; however, it increased the migration ability of both types of cell lines. Importantly, our data suggest that SVIP may increase p53 protein levels in MCF7 cells by inhibiting Hrd1-mediated p53 degradation. Overall, our data reveal the differential expression and function of SVIP on breast cancer cell lines together with in silico data analysis.

Immunogenicity of a xenogeneic multi-epitope HER2+ breast cancer DNA vaccine targeting the dendritic cell restricted antigen-uptake receptor DEC205.

Breast cancer was ranked first in global cancer incidence in 2020, and HER2 overexpression in breast cancer accounts for 20-30% of breast cancer patients. Current therapeutic strategies increase the survival rate, but resistance to them occurs frequently, and there is an urgent need to develop novel treatments such as DNA vaccines which can induce a specific and long-lasting immune response against HER2 antigens. To enhance the immunogenicity of DNA vaccines, dendritic cells (DCs) can be targeted using multi-epitope proteins that provide accurate immune focusing. For this purpose, we generated a DNA vaccine encoding a fusion protein composed of 1) in silico discovered antigenic epitopes of human and rat HER2 proteins (MeHer2) and 2) a single-chain antibody fragment (ScFv) specific for the DC-restricted antigen-uptake receptor DEC205 (ScFvDEC). The xenogeneic multi-epitope DNA vaccine (pMeHer2) encodes three only T-cell epitopes, two only B-cell epitopes, and two T and B cell epitopes, and pScFvDEC-MeHer2 vaccine additionally encodes ScFvDEC introduced at the N terminus of the MeHer2. Then, mouse groups were immunized with pScFvDEC-MeHer2, pMeHer2, pScFvDEC, pEmpty, and PBS to determine the elicited immune response. pScFvDEC-MeHer2 vaccinated mice showed a strong IgG response (P < 0.0001) and pScFvDEC-MeHer2 induced a significant IgG2a increase (P < 0.01). The percentages of both IFN-γ secreting CD4 and CD8 T cells were higher in mice immunized with pScFvDEC-MeHer2 compared with the pMeHer2. pScFvDEC-MeHer2 and pMeHer2 secreted significantly higher levels of extracellular IFN-γ compared with to control groups (P < 0.0001). In addition, the IFN-γ level of the pScFvDEC-MeHer2 vaccine group was approximately two times higher than the pMeHer2 group (P < 0.0001). Overall, this study identified the pScFvDECMeHer2 construct as a potential DNA vaccine candidate, supporting further studies to be conducted on HER2+ animal models.

Novel regulation mechanism of adrenal cortisol and DHEA biosynthesis via the endogen ERAD inhibitor small VCP-interacting protein.

Endoplasmic reticulum-associated degradation (ERAD) is a well-characterized mechanism of protein quality control by removal of misfolded or unfolded proteins. The tight regulation of ERAD is critical for protein homeostasis as well as lipid metabolism. Although the mechanism is complex, all ERAD branches converge on p97/VCP, a key protein in the retrotranslocation step. The multifunctionality of p97/VCP relies on its multiple binding partners, one of which is the endogenous ERAD inhibitor, SVIP (small VCP-interacting protein). As SVIP is a promising target for the regulation of ERAD, we aimed to assess its novel physiological roles. We revealed that SVIP is highly expressed in the rat adrenal gland, especially in the cortex region, at a consistently high level during postnatal development, unlike the gradual increase in expression seen in developing nerves. Steroidogenic stimulators caused a decrease in SVIP mRNA expression and increase in SVIP protein degradation in human adrenocortical H295R cells. Interestingly, silencing of SVIP diminished cortisol secretion along with downregulation of steroidogenic enzymes and proteins involved in cholesterol uptake and cholesterol biosynthesis. A certain degree of SVIP overexpression mainly increased the biosynthesis of cortisol as well as DHEA by enhancing the expression of key steroidogenic proteins, whereas exaggerated overexpression led to apoptosis, phosphorylation of eIF2α, and diminished adrenal steroid hormone biosynthesis. In conclusion, SVIP is a novel regulator of adrenal cortisol and DHEA biosynthesis, suggesting that alterations in SVIP expression levels may be involved in the deregulation of steroidogenic stimulator signaling and abnormal adrenal hormone secretion.

Induction of Divergent Cell Death Pathways by Urea and Carbohydrazide Derivatives.

BACKGROUND: The complexity of cancer biology and the development of chemotherapy resistance are two main obstacles to cancer treatment and necessitate novel anticancer molecules that target different cell death pathways. Modulation of Endoplasmic Reticulum (ER) stress and subsequent activation of the Unfolded Protein Response (UPR) has been proposed as a potential chemotherapeutic target, as prolonged ER stress can lead to cell death via apoptosis or necrosis. OBJECTIVE: The present study aims to evaluate the molecular mechanism underlying the cytotoxic activity of selected urea and carbohydrazide derivatives. METHODS: Cell proliferation assays were performed on HeLa, Capan-1, MCF-7, HCC-1937, and MRC-5 cell lines by WST-1 assay. The expression levels of selected ER stress, autophagy, and apoptosis marker proteins were compared by immunoblotting to characterize the underlying mechanism of cytotoxicity. Flow cytometry was used to detect apoptosis. RESULTS: Of the tested cytotoxic compounds, 3a, 4a, 5a, 6a, and 1b dramatically and 5b moderately increased ER stress-related CHOP protein levels. Interestingly, 5b but not 3a, 4a, 5a, 6a, or 1b increased the expression of proapoptotic proteins such as cleaved PARP-1 and cleaved caspase-3 and -7. The flow-cytometry analysis further confirmed that the cytotoxic activity of 5b but not the other compounds is mediated by apoptosis, demonstrated by a significant increase in the percentage of late apoptotic cells (7-AAD/annexin V double-positive cells). CONCLUSION: Our results suggest that changing a substituent from trifluoromethyl to nitro in urea and carbohydrazide core structure alters the cell death mechanism from apoptosis to an apoptosis-independent cell death pathway. This study shows an example of how such simple modifications of a core chemical structure could cause the induction of divergent cell death pathways.

The molecular and serological investigation of Feline immunodeficiency virus and Feline leukemia virus in stray cats of Western Turkey.

This study aimed to investigate the Feline immunodeficiency virus (FIV) / Feline leukemia virus (FeLV) infection prevalence among looking healthy stray cats in Western Turkey by serologic and molecular-based tests. A total of 1008 blood samples from the stray cats were used in this study. All samples were tested for FIV antibodies / proviral DNA and FeLV antibodies / antigens / proviral DNA. The genetic characterization and phylogenetic analysis of FeLV and FIV were carried out in this study. These cats also tested for Leishmaniasis and Toxoplasmosis previously. FIV Ab and proviral DNA detected in 25.2 % and 25.5 % of samples, respectively. FeLV Ab, Ag, proviral DNA positivity was in 45.2 %, in 3.3 %, in 69.7 %, respectively. The molecular detection and phylogenetic analysis of the current FeLV pol gene and FIV gag gene performed. The molecular characterization for the pol gene of FeLV (enFeLV and exFeLV) among Turkey's cat population was reported for the first time. The exFeLV pol sequences closer to the FeLV-A genotype, and the enFeLV pol sequences overlapped with other enFeLV. The current FIV gag sequences were clustered within the subtypes A, B, and C. The findings revealed FeLV subtype A and FIV subtype-A, subtype-B, subtype-C circulate among Turkish stray cats. Single and multiple co-infection positivity was found higher compared to previous reports.

Investigation of the role of stray cats for transmission of toxoplasmosis to humans and animals living in Izmir, Turkey.

INTRODUCTION: Toxoplasma gondii is a protozoan parasite that has a widespread distribution among mammalians and birds. One of the reasons for the high prevalence may be due to ingesting oocyst disseminated by stray cats' feces. In Turkey, most of the citizens are closely associated with stray cats and they love to pet and feed them on the streets. In this study, we aimed to determine the prevalence of T. gondii DNA in feces of stray cats living in Izmir, Turkey in order to identify the transmission potential to humans and other animals. METHODOLOGY: Feces and blood samples of 465 stray cats were investigated for the presence of T. gondii oocysts by microscopy and for the presence of T. gondii DNA by two real time PCR methods. Furthermore, serum samples were analyzed for anti-T. gondii IgG antibodies using an ELISA. RESULTS: Oocysts were detected in 0.43% of the stray cats by microscopy. T. gondii DNA was detected in 14.37% of the stray cats' feces samples. The seroprevalence rate was 37.84%. In the feces and/or blood PCR positive group, 35.89% of them were seropositive. Among the 176 seropositive cats, T. gondii DNA was detected in feces of 27 cats (15.34%). CONCLUSIONS: This study first time showed the inter relation of T. gondii DNA in feces and blood samples and seropositivity. In sum, over 14% of the stray cats living outdoor may have an important role in transmission of toxoplasmosis to humans in Izmir as well as to other animals.

Toxoplasma gondii destroys Her2/Neu-expressing mammary cancer cells in vitro using a continuous feed medium approach.

INTRODUCTION: Toxoplasma gondii is an opportunistic protozoan and can be grown using several human cell lines. Breast cancer is the second leading cause of cancer death in women. Her2/Neu-expressing mammary cancer cell lines called TUBO can be grown in vitro. In recent years, protozoan parasites have become popular means of use in cancer therapy research. In this study, we analyzed whether T. gondii tachyzoites can destroy TUBO cells using a novel continuous feed medium approach. METHODOLOGY: Two sets of flasks (each containing four groups) containing TUBO cells were inoculated with T. gondii Ankara strain tachyzoites. First set containing 5×106 TUBO cells were inoculated with TUBO-tachyzoite ratios of 1:2, 1:1, 2:1, and 4:1 and second set containing 1×106 TUBO cells were inoculated with TUBO-tachyzoite ratios of 10:1, 100:1, 1000:1, and 2000:1. Thereafter, culture supernatants were harvested at various days until TUBO cells were destroyed and tachyzoites were counted. RESULTS: In the first and second sets of flasks, TUBO cells were destroyed between days 8 to 12 and 12 to 25, respectively. In addition, the amount of tachyzoites increased 7- 43 and 595 to 112500 times in the first and second set of flasks, respectively. CONCLUSIONS: These results show that T. gondii tachyzoites successfully destroy Her2/Neu-expressing mammary cancer cells using a continuous feed medium approach. Although this idea may be too premature for the moment, the approach defined herein may support future researchers investigating the relationship between cancer and parasites which can make important progress toward saving cancer patient lives.

Genotyping of Pneumocystis jirovecii isolates obtained from clinical samples by multilocus sequencing: a molecular epidemiology study conducted in Turkey.

Pneumocystis jirovecii is an opportunistic respiratory pathogen causing Pneumocystis pneumonia (PcP) in immunocompromised patients. The aim of this study was to investigate the genetic diversity of P. jirovecii isolates (n: 84) obtained from PcP patients using multilocus sequencing method based on mt26S, SOD, and CYB loci. Among the 84 clinical samples that were positive for P. jirovecii DNA, 31 (36.90%) of them were genotyped using at least one locus. Of the 31 clinical samples, 26 of them were successfully genotyped using all loci whereas three samples were genotyped using either mt26S/CYB loci or mt26S/SOD loci. Additionally, there were two more clinical samples that were genotyped using CYB or SOD locus. Using mt26S locus, genotypes 2, 3, 7, and 8 were detected. Frequencies of genotype 7 and 8 were higher and both of them were found in 11 (n: 29; 37.93%) clinical samples. Using SOD locus, SOD 1, 2, and 4 genotypes were detected. SOD 1 was the predominant genotype (20/28; 71.42%). During the analyses of CYB locus, CYB 1, 2, 5, 6, and 7 as well as a new CYB genotype were detected. CYB 1 (16/29; 55.17%) and 2 (10/29; 34.48%) were the predominant genotypes. Overall, according to the multilocus sequencing results E, F, M, N, P, and V multilocus genotypes were detected among the PcP patients. In addition, SOD 1 was the predominant genotype and CYB had a more polymorphic locus.

The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology.

Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. In the laboratory diagnosis of toxoplasmosis, serological tests have importance in detecting antibody response. Traditionally T. gondii tachyzoites grown in vivo are being used as an antigen source in serological assays. Currently, tachyzoites produced in vitro are being tested as an antigen source in order to decrease animal use. Microcarrier technology allowed us to grow anchorage-dependent host cells on microcarrier suspension in short time and approximately 10 times more than traditional flask technique. The ability of T. gondii tachyzoites to grow in host cells adhered to microcarriers has not been analyzed yet. In this study, we aimed to develop a novel in vitro culture method to produce T. gondii tachyzoites abundantly using HeLa cells adhered to Cytodex 1 microcarriers. Initially, the growth of HeLa cells adhered to Cytodex 1 was analyzed using RPMI 1640, DMEM, and EMEM. Next, HeLa cells with a concentration of 1 × 105 cells/ml and 2 × 105 cells/ml were adhered to Cytodex 1 and grown in spinner flasks. Then, T. gondii tachyzoites were inoculated with 1:1 and 2:1 cell:tachyzoite ratios to HeLa cells adhered to microcarriers in spinner flaks. During continuous production in spinner flasks, tachyzoites were harvested at the 2nd, 4th, and 7th day of culture and the quality of antigens produced from these tachyzoites were tested in ELISA and Western Blotting using sera of patients with toxoplasmosis. The optimization studies showed that finest HeLa inoculation value was 2 × 105 cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 × 107) was 1:1 at the 4th day of inoculation. According to the results of ELISA comparing HeLa cell and mouse derived antigens, the highest correlation with mouse antigen was achieved at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio (P < 0.0001). The sensitivity and specificity ratios of ELISA were 100%. In addition, Western blotting banding patterns of the antigen derived at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio was comparable with mouse derived antigen. Overall, this novel methodology can be an alternative source of antigen in diagnostic assays, decrease animal use for antigen production, and contribute to the solution of ethical and economic problems.

Detection of Toxoplasma gondii in a Eurasian Badger (Meles meles) Living in Wildlife Areas of Izmir, Turkey.

Toxoplasma gondii is an obligatory intracellular protozoon parasite that causes toxoplasmosis in humans and all warm-blooded animals. In this study, we aimed to investigate the presence of T. gondii DNA in a Eurasian badger (Meles meles) that was found dead in the wildlife area of Izmir. According to the results of real time polymerase chain reaction, T. gondii REP gene was found to be positive in the Eurasian badger brain homogenate. In conclusion, Eurasian badger, a known carnivore, can be a potential source of toxoplasmosis in the natural settings of Izmir, Turkey.

Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey.

Toxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype(s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of Izmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondii strains within type II and III lineage have close relation with strains previously isolated from stray cats in Izmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation.

[The importance of the contribution of rapid test, serological and molecular methods in the diagnosis of two imported malaria cases with atypical microscopy]. / Mikroskopide atipik görünümlü dis kaynakli iki sitma olgusunda hizli test, serolojik ve moleküler yöntemlerin taniya katkisinin önemi.

Malaria is a widespread and life-threatening disease in tropical and subtropical regions. In patients with typical clinical symptoms, malaria is considered as a preliminary diagnosis if there is a travel history to malaria-endemic areas. The basis of the laboratory diagnosis of malaria is the microscopic examination of Giemsa stained smears. On the other hand, the diagnosis and differentiation of Plasmodium species with microscopic examination may have some difficulties. In the first case, adifferent appearance from the classical Plasmodium vivax erythrocytic forms in infected erythrocytes were detected in 1% of all erythrocytes in thin smear blood preparations of a 26-year-old male with complaints of fever and chills and a story of travel to Nigeria. It was observed that parasitic nuclei were not prominent, and were located in the cytoplasm irregularly as chromatin or dye particles, nucleus fragments similar to Schüffner's granules in the form of scattered and granular spots were present in some erythrocytes, the cytoplasm of some Plasmodium erythrocytic forms were irregular and nuclei were not seen. There were no Schüffner's granules in any of the infected erythrocytes. P.vivax was detected by the rapid diagnostic test (OptiMAL, DiaMed GmbH, Switzerland), which searches for the antigens of Plasmodium species, in the peripheral blood sample of the patients. The P.vivax 18S rRNA gene was also detected by the multiplex real-time polymerase chain reaction. Antibodies against Plasmodium species were searched by using the Pan Malaria Antibody CELISA (CeLLabs Pty Ltd, Brookvale, Australia) kit in the patient's serum sample and the optical density (OD) value of the patient sample was measured five times the OD value of the positive control. In the second case, adifferent appearance from the classical P.falciparum erythrocytic forms in infected erythrocytes were detected in 12% of all erythrocytes in thin smear blood preparations of a 31-year-old male who has been suffering from persistent fever, severe headache, pain in the eyes and was known to be working in Nigeria. It was observed that some Plasmodium trophozoites have 1/3 of the size of erythrocytes such as P.vivax and have non-granular cytoplasm, some erythrocytic forms were round and the nucleus and cytoplasm were hardly distinguished, some of them were seen as crescent and close to the nucleus of the cytoplasm and some erythrocytic forms had characteristically a single nucleus and a scattered cytoplasm, similar to mature trophozoites of P.vivax. Although the Plasmodium young trophozoites were similar to P.vivax in means of magnitude, the forms in which the nuclei adhered to the erythrocyte wall were common. There were no P.falciparum gametocyte forms. P.falciparum like young trophozoite was observedonly in one of the four smears. P.falciparum was detected by the commercial rapid diagnostic test and P.falciparum 18S rRNA gene was also detected by the multiplex real-time polymerase chain reaction. Antibody formation against Plasmodium species was not detected in the ELISA test. In these case reports, the importance of the support of rapid diagnostic tests, serological and molecular methods to microscopic diagnosis and species determination of two imported malaria cases were demonstrated.

Screening of immunocompromised patients at risk of strongyloidiasis in western Turkey using ELISA and real-time PCR.

BACKGROUND/AIM: Strongyloides stercoralis causes life-threatening hyperinfection or disseminated strongyloidiasis in immunocompromised patients such as HIV-positive, organ transplantation, and cancer patients. This study investigated the presence of strongyloidiasis in immunocompromised patients for the first time in Turkey. MATERIALS AND METHODS: Serum and stool samples were collected from 108 patients (25.9% of them were chronic renal failure and 74.1% were renal transplantation patients) who were admitted to Ege University Medical School in Izmir, located in western Turkey. Serum samples were analyzed by ELISA (DRG, Germany) and the presence of 18S rRNA gene of S. stercoralis was detected in stool samples by real-time PCR. RESULTS: The analysis of serum samples showed that only one patient was anti-S. stercoralis IgG antibody and real-time PCR positive (0.92%). The patient was treated twice with albendazole (400 mg/day for 3 days) at 2-week intervals. Follow up real-time PCR was negative and the patient became seronegative 6 months after the initial diagnosis. CONCLUSION: This screening showed that the prevalence of strongyloidiasis in this small group of patients who were at risk of strongyloidiasis was 0.92%. Overall, the results showed that more systematic studies are required in Turkey to show the prevalence of strongyloidiasis.

Diagnosis and Species Discrimination of Positive Malaria Samples by Real-Time Polymerase Chain Reaction.

OBJECTIVE: Malaria is an important tropical disease that is detected in 198 million people and causes 367-755 thousand deaths annually. Recently, the real-time Polymerase Chain Reaction (PCR) technique has enabled quick determination of Plasmodium spp. and species identification in the same assay with a low contamination risk. In the present study, we aimed to use real-time PCR targeting the 18S rRNA gene to diagnose Plasmodium spp. and perform species identification. METHODS: DNA samples of 15 patients with malaria (14 caused by P. vivax, 1 caused by P. falciparum) confirmed by microscopy as well as positive control plasmids were used. As the negative control, DNA samples of 15 individuals without malaria were used. RESULTS: According to the results of real-time PCR, samples of 15 patients with malaria were found to be positive for Plasmodium spp. Melting curve analysis showed that 14 of them were P. vivax and the remaining was P. falciparum. In addition, mixed infection with P. falciparum and P. vivax was successfully detected by real-time PCR when DNA of P. falciparum- and P. vivax-positive samples was experimentally mixed. CONCLUSION: The present study showed that real-time PCR can be useful in the diagnosis and species identification of Plasmodium spp. as well as the detection of mixed infections in addition to microscopy in Turkey.

[Detection of Echinococcus granulosus and Echinococcus multilocularis in cyst samples using a novel single tube multiplex real-time polymerase chain reaction]. / Kist örneklerinde yeni bir tek tüp multipleks gerçek zamanli polimeraz zincir reaksiyonu ile Echinococcus granulosus ve Echinococcus multilocularis'in saptanmasi.

Cystic echinococcosis (CE) and alveolar echinococcosis (AE) caused by Echinococcus granulosus and Echinococcus multilocularis, respectively, are important helminthic diseases worldwide as well as in our country. Epidemiological studies conducted in Turkey showed that the prevalence of CE is 291-585/100.000. It has also been showed that the seroprevalence of AE is 3.5%. For the diagnosis of CE and AE, radiological (ultrasonography, computed tomography, magnetic resonance) and serological methods, in addition to clinical findings, are being used. The definitive diagnosis relies on pathological examination When the hydatid cysts are sterile or does not contain protoscolex, problems may occur during pathological discrimination of E.granulosus and E.multilocularis species. In this study, we aimed to develop a novel multiplex real-time polymerase chain reaction (M-RT-PCR) targeting mitochondrial 12S rRNA gene of E.granulosus and E.multilocularis using Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') and Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primers and three different probes; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fluoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTTGGTGTAGTAGTTGAGATT-phosphate-3') that will enable the diagnosis of CE and AE in same assay. During M-RTR-PCR, plasmids containing E.granulosus (GenBank: AF297617.1) and E.multilocularis (GenBank: NC_000928.2) mitochondrial 12S rRNA regions were used as positive controls. Cysts samples of patients which were pathologically confirmed to be CE (n: 10) and AE (n: 15) and healthy human DNA samples (n: 25) as negative control as well as DNA samples of 12 different parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) were used to develop M-RT-PCR. E.granulosus and E.multilocularis control plasmids were constructed to detect analytic sensitivity of the test using TOPO cloning. Positive control plasmids were diluted to determine analytical sensitivity and specificity by distilled water at 10(6)-10(5)-10(4)-10(3)-10(2)-10(1)-1 plasmid copy of dilution in each reaction. According to the results, analytical sensitivity of the assay for E.granulosus and E.multilocularis was 1 copy plasmid/µl reaction. The non-existence of cross reactivity with 12 different parasites' DNA samples showed the analytical specificity of the assay. Displaying Echinococcus DNA in cyst samples among 25 patients and species discrimination as well as non-existence of cross reactivity with human DNA samples showed that the clinical sensitivity and specificity of the assay were 100%. As a result, the M-RT-PCR developed in the present study provided a sensitive, specific, rapid, and reliable method in the diagnosis of echinococcosis and the discrimination of E.granulosus and E.multilocularis from cyst samples.