Determination of Species of Leishmania Using HSP70 Gene in Patients Referred to Selected Health Centers of Iran

Background: Cutaneous leishmaniasis is caused by Leishmania major and Leishmania tropaica. Iran is considered as one of the world's largest leishmaniosis centers. In this study, the restriction fragment length polymorphism (RFLP)-PCR method was used to determine the species of pathogenic parasites. Materials and Methods: In this cross-sectional study, 120 patients with suspected cutaneous ulcer referring to the selected medical centers of Shahid Beheshti University of Medical Sciences were selected. Their demographic characteristics were recorded. After diagnosis of parasite by microscopy, positive samples were cultured in NNN medium. After DNA extraction of cultured parasite, PCR was performed for amplification of the HSP70 gene. The PCR products were digested with HaeIII restriction enzyme; the obtained patterns were compared with the same genes in the GenBank database. Results: A 1422 base pair fragment was detected in PCR of HSP70 gene in 30 positive samples. After digestion, 16 (53.3%) of the samples, had an enzymatic digestive pattern compatible to L. major and 14 (46.7%) others had the L. tropica profile. More information concerning demographic aspects were obtained after analyses them with the infected samples. Conclusion: Regarding the history of patient trips to their hometown, the transfer does not appear to have occurred in areas covered by the university's medical centers, but the presence of these patients in these areas requires careful monitoring them. Additionally, control of population and mosquito species is needed. The results of this study sustain that the HSP70 gene still has sufficient ability to differentiate between two different species of L. major and L. tropica

Optimization of Real-Time Quantitative PCR assay for detection of Echinococcus granulosus in fecal samples

Background: Echinococcosis/hydatidosis is one of the most important zoonotic diseases. The definitive hosts for Echinococcus granulosus (E. granulosus) include a wide variety of the Canidae and dogs. Early detection of Echinococcosis in dogs is the most influential factor in improving the prevention and control of hydatidosis. The primary purpose of the present study was to optimize the real-time quantitative PCR to diagnose E. granulosus infection in dogs before the disposal of eggs.Materials and Methods: Three puppies were selected to be inoculated by 70000 protoscoleces. Normal saline was inoculated to the other two puppies chosen as an experimental negative control group. Ten privately owned healthy puppies were selected for the natural negative control group. Stool samples were collected on days 7, 14, 21, 28, and 35 post-infection, DNA was extracted, then a 287bp fragment of tandem repeat region gene was amplified and cloned into linearized TA vectors. Serial dilutions of recombinant plasmid DNA and a standard curve were established. The copy amount of DNA in each sample was determined based on the standard curve.Results: The minimum time copro –DNA could be detected in the stool sample was found on the 7th day post-infection, which was equal to 9750×10-8 copy number or 9.75 pg of DNA. The assay was linear in 105 to 108 copies of the recombinant plasmid per microliter.Conclusion: The real-time PCR assay diagnosed the infection eight days earlier than the copro antigen ELISA method (7 days versus 15 days, respectively).Conclusion: The real-time PCR assay diagnosed the infection eight days earlier than the copro antigen ELISA method (7 days versus 15 days, respectively)

An Experimental Model of Primary Amoebic Meningoence phalitis Due to Naegleria australiensis in Iran

Background: The main aim of the present research was to develop the experimental meningo encephalitis due to Naegleria australiensis isolated from geothermal water sources in mice model, November 2017 in Iran. Methods: Naegleria australiensis was isolated from geothermal water sources in northern Iran. The number of amoebae was adjusted to be 1×104/ml amoebae. The experimental infection was done using 3 wk old male (BALB/c) mice. Seven animals were used for experimental amebic infection and one animal was selected for the control. Intranasal (IN) and intracerebral (IC) inoculation of amoebae were done. The mice were then monitored on daily observation and as soon as they present any brain involvement they sacrificed. The brain of all animals was then dislocated and passaged in non-nutrient agar. Results: One mouse out of seven infected mice were showed clinical symptoms of meningoencephalitis. Within few hours of culture of the brain, many vegetative forms of amoebae were detected in plate culture. The other infected animals and control mice showed no clinical symptoms until day 14. After 14 d all the animals sacrificed. The culture was negative up to one month. Conclusion: The lack of brain involvement of other animals in the present study could be due to animal immune system or it may be possible that the amoebae did not reach to olfactory bulb of nostrils

Study of MazEF, sam, and phd-doc putative toxin–antitoxin systems in Staphylococcus epidermidis

Today, to replace the antibacterial targets to overcome antibiotic resistance, toxin–antitoxin (TA) system is noticeable, where the unstable antitoxin neutralizes the stable toxin and protects the bacteria against the toxic effects. The presence and expression of TA genes in clinical and non-clinical strains of Staphylococcus epidermidis were investigated in this study. After identification of three TA pairs (mazEF, sam, and phd-doc) via existing databases (earlier, there has been no information in the case of S. epidermidis isolates), the presence and expression of these pairs were investigated by PCR and q-PCR, respectively. We detected three TA modules in all antibiotic sensitive and resistant isolates. In addition, q-PCR analysis revealed that the transcripts were produced from the three TA modules. This study showed the significant prevalence of these systems in pathogenic bacteria and they were equally found in both oxacillin-resistant and oxacillin-susceptible bacteria. The high prevalence of three systems can make them suitable as potential targets for antibiotic therapy