Epidemiology of yeast species causing bloodstream infection in Tehran, Iran (2015-2017); superiority of 21-plex PCR over the Vitek 2 system for yeast identification.

Introduction. Given the limited number of candidaemia studies in Iran, the profile of yeast species causing bloodstream infections (BSIs), especially in adults, remains limited. Although biochemical assays are widely used in developing countries, they produce erroneous results, especially for rare yeast species.Aim. We aimed to assess the profile of yeast species causing BSIs and to compare the accuracy of the Vitek 2 system and 21-plex PCR.Methodology. Yeast blood isolates were retrospectively collected from patients recruited from two tertiary care training hospitals in Tehran from 2015 to 2017. Relevant clinical data were mined. Identification was performed by automated Vitek 2, 21-plex PCR and sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2).Results. In total, 137 yeast isolates were recovered from 107 patients. The overall all-cause 30-day mortality rate was 47.7 %. Fluconazole was the most widely used systemic antifungal. Candida albicans (58/137, 42.3 %), Candida glabrata (30/137, 21.9 %), Candida parapsilosis sensu stricto (23/137, 16.8 %), Candida tropicalis (10/137, 7.3 %) and Pichia kudriavzevii (Candida krusei) (4/137, 2.9 %) constituted almost 90 % of the isolates and 10 % of the species detected were rare yeast species (12/137; 8.7 %). The 21-plex PCR method correctly identified 97.1 % of the isolates, a higher percentage than the Vitek 2 showed (87.6 %).Conclusion. C. albicans was the main cause of yeast-derived fungaemia in this study. Future prospective studies are warranted to closely monitor the epidemiological landscape of yeast species causing BSIs in Iran. The superiority of 21-plex PCR over automated Vitek 2 indicates its potential clinical utility as an alternative identification tool use in developing countries.

Leishmania major p27 gene knockout as a novel live attenuated vaccine candidate: Protective immunity and efficacy evaluation against cutaneous and visceral leishmaniasis in BALB/c mice.

Leishmaniasis is a growing health problem in many parts of the world and efforts to find vaccine against the disease are a public health priority. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania spp. Defined genetic alteration of the Leishmania genome can be achieved using a gene-targeted disruption strategy that allows for the selection of parasites lacking genes essential for long-term survival and virulence. Previously, we demonstrated that genetically modified live attenuated Leishmania major, lacking the p27gene (Lmp27-/-) is safe and induces cellular immunity in BALB/c mice. p27 is a component of the COX complex that is responsible for ATP synthesis. In the current study, the Lmp27-/- strain was assessed as a live attenuated vaccine. Overall protective immunity and efficacy were evaluated at various time periods following Leishmania major (L. major) and Leishmania infantum (L. infantum) challenges separately in BALB/c mice. Cytokine and anti-Leishmania antibody levels, splenocyte proliferation, delayed type hypersensitivity (DTH), skin lesion development, and parasite burden in the liver and spleen were the measured variables. The results demonstrated that immunized mice had a significant T-helper type 1 (Th1) response, smaller skin lesions and lower parasite burdens in their liver and spleens following a L. major challenge. Furthermore, the Lmp27-/- mutant also granted cross-protection against L. infantum infection. These results suggest that immunization with Lmp27-/- parasites provide significant protective immunity and efficacy against infection with homologous as well as heterologous species of Leishmania parasites.

Comparison of p27 Gene Expression of Promastigote and Amastigote Forms of Leishmania major (MRHO/IR/75/ER) by Real-time RT-PCR.

BACKGROUND: Cutaneous leishmaniasis (CL) is one of the world health problems. Leishmania major is the etiological agent of zoonotic cutaneous leishmaniasis (ZCL). Promastigote and amastigote are two morphological forms of Leishmania parasites that express different proteins and p27 is an important gene encoding cytochrome c oxidase (COX) component. P27 gene expresses a 27 kDa protein that essential in ATP synthesis. This study aimed to compare p27 gene expression in promastigote and amastigote forms in Iranian strain of L. major (MRHO/IR/75/ER). METHODS: This study was conducted in 2015. Clinical isolates of CL patients from north, center, west and south parts of Iran were collected and identified by PCRRFLP. After RNA extraction of promastigotes and amastigotes and cDNA synthesis, the expression level of p27 gene was compared by real-time RT-PCR. RESULTS: By comparison of expression level between amastigote and promastigote forms of Iranian strain of L. major, up-regulation of p27 gene (2.73 fold) was observed in amastigotes. Moreover, there was no significant difference in p27 gene expression between L. major isolates. CONCLUSION: p27 gene and protein can be considered as a target in recombinant vaccine production and treatment process.

Development of a new live attenuated Leishmania major p27 gene knockout: Safety and immunogenicity evaluation in BALB/c mice.

Genetically modifying Leishmania major by eliminating essential virulence genes have been proposed as potential vaccine candidates. p27 is a COX component that is responsible for ATP synthesis. In this study a new mutant of Leishmania major (L. major) (MRHO/IR/75/ER) lacking the p27 gene (Lmp27-/-) was produced via homologous recombination, marking the first time such a strain has been developed. In vitro macrophage infectivity and In vivo safety, and overall immunogenicity were evaluated at various time periods following inoculation into BALB/c mice. Skin lesion development, parasite burden in the liver and spleen, cytokine and antibody levels, splenocyte proliferation, and delayed type hypersensitivity (DTH) were the measured variables. Results demonstrated that the Lmp27-/- mutant caused no skin lesion, had low parasitic burdens in the liver and spleen, and had a significantly increased Th1 response. These results suggest that the Lmp27-/- mutant has the potential to be evaluated as a vaccine candidate.

Effect of biogenic selenium nanoparticles on ERG11 and CDR1 gene expression in both fluconazole-resistant and -susceptible Candida albicans isolates.

BACKGROUND AND PURPOSE: Candida albicans is the most common Candida species (sp.) isolated from fungal infections. Azole resistance in Candida species has been considerably increased in the last decades. Given the toxicity of the antimicrobial drugs, resistance to antifungal agents, and drug interactions, the identification of new antifungal agents seems essential. In this study, we assessed the antifungal effects of biogenic selenium nanoparticles on C. albicans and determined the expression of ERG11 and CDR1 genes. MATERIALS AND METHODS: Selenium nanoparticles were synthesized with Bacillus sp. MSH-1. The ultrastructure of selenium nanoparticles was evaluated with a transmission electron microscope. The antifungal susceptibility test was performed according to the modified Clinical and Laboratory Standards Institute M27-A3 standard protocol. The expression levels of the CDR1 and ERG11 genes were analyzed using the quantitative real-time polymerase chain reaction (PCR) assay. RESULTS: The azole-resistant C. albicans and wild type C. albicans strains were inhibited by 100 and 70 µg/mL of selenium nanoparticle concentrations, respectively. The expression of CDR1 and ERG11 genes was significantly down-regulated in these selenium nanoparticle concentrations. CONCLUSION: As the findings indicated, selenium nanoparticles had an appropriate antifungal activity against fluconazole-resistant and -susceptible C.albicans strains. Accordingly, these nanoparticles reduced the expression of CDR1 and ERG11 genes associated with azole resistance. Further studies are needed to investigate the synergistic effects of selenium nanoparticles using other antifungal drugs.