Isolation of Crithidia spp. from lesions of immunocompetent patients with suspected cutaneous leishmaniasis in Iran.

OBJECTIVE: Leishmania major has been considered as the main aetiological agent of cutaneous leishmaniasis in Iran. However, there are recent reports about the existence of Crithidia spp in cutaneous lesions in southern Iran. Therefore, this study was designed to decipher some morphological, biological and molecular aspects of this phenomenon. METHODS: Clinical isolates were obtained from 167 patients with cutaneous ulcers. A set of specific primers based on GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) gene were used to distinguish between Crithidia and Leishmania genera. For molecular analysis, Pulsed Field Gel Electrophoresis and Mi-Seq Illumina platform were applied. Then, morphological analysis and some biological features (including potential growth at 37 °C and the ability of infecting mammalian macrophages) were studied. RESULTS: In 92.8% of clinical cases, L. major was the only causative microorganism isolated; in 5.4% of cases, co-infection of L. major and Crithidia spp. was identified; and in 1.8% of lesions, only Crithidia spp. were found. CONCLUSION: We isolated Crithidia spp. from clinical samples of patients suspected of cutaneous leishmaniasis in Iran, indicating that Crithidia spp. are capable of surviving at human body temperature and infecting macrophage cells. This raises questions on the influence of this phenomenon on pathogenicity, therapeutic outcome and disease control strategies.

Lower Abundance and Impaired Function of CD71+ Erythroid Cells in Inflammatory Bowel Disease Patients During Pregnancy.

BACKGROUND AND AIMS: CD71+ erythroid cells are enriched during pregnancy with immuno suppressive properties. We investigated the frequency and functionality of CD71+ erythroid cells in peripheral blood, cord blood, and placenta of inflammatory bowel disease [IBD] patients versus healthy controls [HCs]. We aimed to determine their role in IBD pathogenesis during pregnancy. METHODS: Peripheral blood was collected at preconception, the first, second and third trimesters, and postpartum. Cord blood and placental tissues were collected at the time of birth. Cells from different specimens were subjected to immune-phenotyping and functional assays. CD71+ erythroid cells were purified for quantitative polymerase chain reaction [qPCR] analysis. Using an allogeneic mouse model of pregnancy, the effects of CD71+ erythroid cells depletion on intestinal homeostasis and dysbiosis was studied. RESULTS: IBD patients had lower CD71+ erythroid cells during pregnancy compared with HCs. Placenta and cord blood CD71+ erythroid cells from IBD patients exhibited impaired functionality and expressed lower inhibitory molecules including VISTA, TGF-ß, and reactive oxygen species [ROS]. Lower CD71+ erythroid cells were correlated with reduced regulatory T cells and increased immune-activation in IBD patients. Depletion of CD71+ erythroid cells in an allogeneic pregnancy model resulted in upregulation of TLRs, IL-6, and CXCL-1, and enhanced production of TNF-α, in intestinal tissues. In contrast, TGF-ß gene expression was reduced. Excessive inflammatory response in the gut [e.g. TNF-α] affects intestinal integrity and CD71+ erythroid cells impact on the gut's bacterial composition. CONCLUSIONS: Reduced frequency and/or impaired functionality of CD71+ erythroid cells during pregnancy may predispose IBD patients to a more pro-inflammatory milieu in their gastrointestinal tract, characterised by lower Tregs, higher IL-6, and TNF-α, and dysbiosis.

Evaluation of Correlation between the In Vitro Susceptibility of Field Isolates of Leishmania major and Clinical Outcomes of Meglumine Antimoniate Therapy in Fars Province, Iran.

BACKGROUND: This study was designed to detect whether there is a correlation between in vitro susceptibility of field isolates of Leishmania major and the clinical outcomes of meglumine antimoniate (Glucantime®) therapy, the mainstay of cutaneous leishmaniasis treatment in Iran. METHODS: Forty-three patients infected with L. major were enrolled in this study from October 2009 to March 2010 and categorized as responsive or unresponsive to Glucantime® treatment after receiving the appropriate therapy. Then, intracellular amastigote approach was conducted on these field strains to investigate in vitro drug susceptibility as well. RESULTS: At clinical level, out of 43 patients, 15 were clinically non-responsive and 28 were responsive to antimony therapy. All those 28 clinically sensitive strains were susceptible to antimony in the in vitro assay, whereas merely 11 isolates from 15 non-healing isolates were resistant in vitro. Finally, a good correlation (78.9%) with high sensitivity, specificity (100/73) between clinical outcomes and the in vitro susceptibility test was achieved. CONCLUSION: The intracellular amastigote model could be an appropriate assay for evaluation of the in vivo drug sensitivity of field isolates. However, more comprehensive studies with larger sets of isolates are needed to confirm these preliminary data.

Expression Analysis of Multiple Genes May Involve in Antimony Resistance among Leishmania major Clinical Isolates from Fars Province, Central Iran.

BACKGROUND: Treatment of Cutaneous Leishmaniasis (CL) is being faced with serious difficulties in Fars Province, due to emerging of resistance against meglumine antimonite (Glucantime®). In this context, determining some biomarkers for drug sensitivity monitoring seems to be highly essential. Different studies have been carried out to decipher the genes might be involved in antimony resistant phenotype in Leishmania spp. Here, we selected three genes: AQP (as drug transporter), TDR-1-1(as drug activator), and γ-GCS (inducing reduction environment) for comparative expression analysis on clinical resistant and sensitive isolates of L. major. METHODS: The clinical isolates of L. major were collected from CL patients referred to Valfajr Health Center, Shiraz from Oct 2011 to Feb 2012. The susceptibility test was performed to confirm drug sensitivity of strains in vitro as well. Then, the gene expression analysis was performed by quantitative real-time PCR using SYBR® Green. RESULTS: By comparison of expression level between strains, up regulation of γ-GCS gene and down regulation of AQP gene were observed in resistant strains compared to the sensitive isolates; however, down regulation of AQP was not statistically specific. Analysis of TDR-1-1 gene unexpectedly showed a high level of expression in the non-responsive cases. CONCLUSION: The γ-GCS, at least, can be considered as a suitable molecular marker for screening antimony sensitivity in clinical isolates, although AQP and TDR-1-1gene seem not to be reliable resistant markers.