Characterization of bacteria expectorated during forced salivation of the Phlebotomus papatasi: A neglected component of sand fly infectious inoculums.

The infectious inoculum of a sand fly, apart from its metacyclic promastigotes, is composed of factors derived from both the parasite and the vector. Vector-derived factors, including salivary proteins and the gut microbiota, are essential for the establishment and enhancement of infection. However, the type and the number of bacteria egested during salivation is unclear. In the present study, sand flies of Phlebotomus papatasi were gathered from three locations in hyperendemic focus of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan Province, Iran. By using the forced salivation assay and targeting the 16S rRNA barcode gene, egested bacteria were characterized in 99 (44%) out of 224 sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. Ten top genera of Spiroplasma, Ralstonia, Acinetobacter, Reyranella, Undibacterium, Bryobacter, Corynebacterium, Cutibacterium, Psychrobacter, and Wolbachia constituted >80% of the saliva microbiome. Phylogenetic analysis displayed the presence of only one bacterial species for the Spiroplasma, Ralstonia, Reyranella, Bryobacter and Wolbachia, two distinct species for Cutibacterium, three for Undibacterium and Psychrobacter, 16 for Acinetobacter, and 27 for Corynebacterium, in the saliva. The abundance of microbes in P. papatasi saliva was determined by incorporating the data on the read counts and the copy number of 16S rRNA gene, about 9,000 bacterial cells, per sand fly. Both microbiological and metagenomic data indicate that bacteria are constant companions of Leishmania, from the intestine of the vector to the vertebrate host. This is the first forced salivation experiment in a sand fly, addressing key questions on infectious bite and competent vectors.

Comparison of the Diagnostic Performance of Antigen B Purified from Sheep Hydatid Cyst Fluid (HCF) with Commercial ELISA Kit.

INTRODUCTION: Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode of Echinococcus granulosus. CE is a health problem in Middle Eastern countries, such as Iran. The purpose of this study was to purify subunit 8 KDa antigen B from crude sheep hydatid cyst fluid (HCF) and compare its sensitivity and specificity with a commercial human ELISA kit (PT-Hydatid-96). METHODS: 28 sera samples were collected from hydatid cyst patients who had surgery for a hydatid cyst and had their disease confirmed by pathology after the surgery. Furthermore, 35 samples of healthy individuals with no history of hydatid cysts were collected, as were nine serum samples from parasite-infected non-CE patients. HCF was obtained from sheep fertile cysts at a Sari slaughterhouse and used as an antigen. In an indirect ELISA test, the B antigen was employed, and the results were compared to those from a commercial ELISA kit. RESULTS: The results of this study were analyzed using the Kappa test. The commercial ELISA kit showed 17 cases (23.6%) positive, 44 cases (61.1%) negative, and 11 cases (15.3%) borderline. B antigen showed that 18 (25%), 43 (59.7 %), and 11 (15.3%) were positive, negative, and borderline, respectively. One sample (1.4% of 72 total samples) of 35 serum samples from healthy individuals was positive using B antigen-based ELISA. In addition, all nine serum samples from parasite-infected non-CE patients were negative for both tests. The sensitivity and specificity of the commercial ELISA kit have been evaluated at 60.7% and 100%, respectively. For B antigenbased ELISA, these values are 64.3 and 97.7%, respectively. CONCLUSION: Antigen B produced from hydatid cyst fluid is a promising option for serological identification of hydatid cysts in both infected and healthy individuals. In an indirect ELISA test, hydatid fluid antigen could be used as a precise source of detection.

Type-II progressive censoring with GLM-based random removal mechanism dependent on the experimental conditions.

This article presents a novel stochastic removal mechanism under Type-II progressive random censoring in which removal probabilities are allowed to be dependent on the lifetime conditions through Generalized Linear Models (GLM). These conditions potentially include failure distances (the time required to observe the next failure) or other covariate information available in the experiment. The proposed GLM-based random removal mechanism includes a set of tuning parameters that are determined by the researcher according to the possible failure distance category. These parameters allow flexible determination of the removal probabilities leading to necessary experimental cost and time reductions. To establish the proposed mechanism, the Proportional Hazard Rate (PHR) family of distributions is considered. Also, the maximum likelihood estimators of parameters and their asymptotic variances are derived for the Weibull distributed lifetime data. A simple simulation algorithm for generating Type-II progressive censoring samples with GLM-based dependent removal probabilities is also presented. The expected experiment time required to complete the life test under this censoring scheme is also investigated using the Monte Carlo integration method. Several simulation studies are conducted to evaluate and compare the performance of the proposed mechanism. A sensitivity analysis is also considered to study the effect of misspecification of removal mechanism coefficients. Finally, two real data sets are analyzed for illustrative purposes.

Involvement of tryparedoxin peroxidase (TryP) and trypanothione reductase (TryR) in antimony unresponsive of Leishmania tropica clinical isolates of Iran.

Clinical resistance to pentavalent antimonial compounds has long been recognized as a major problem in the treatment of human leishmaniasis. Trypanothione metabolism, the main form of thiol, has shown to play a central role in antimony resistance of laboratory-generated resistant Leishmania spp. and field-isolated resistant L. donovani; but the mechanism of antimony resistance in the clinical isolates of L. tropica causing anthroponotic cutaneous leishmaniasis (ACL) is less studied. Patients were selected among confirmed positive ACL cases who referred to Pasteur Institute of Iran, Tehran, from endemic regions of north-east and south of Iran. L. tropica clinical isolates were collected from patients who were either treatment-responsive (MAS=S1 to S5) or unresponsive (MAR=R1 to R4) to Glucantime® (meglumine antimoniate=MA). Isolates were tested for sensitivity to trivalent antimony (SbIII) in promastigotes and to pentavalent antimony (SbV) in intracellular amastigotes stages. Intracellular thiol levels were assayed and trypanothione-dependent components, including trypanothione reductase (TR) and tryparedoxin peroxidase I (TryP) were analysed at protein level and enzymatic activity in isolates. The MAR isolates had an approximate two fold increase in the levels of intracellular thiols (P< 0.05) accompanied by an average 5-10 fold increase in in vitro resistance to antimony. TryP was amplified at the protein level in all MAR strains as compared to the MAS strains (range: 2.8-5.6 fold). All MAR isolates metabolized H2O2 at higher rates than MAS isolates (8.55±0.75 nmol/min/mg vs. 3.14±0.36 nmol/min/mg) (P< 0.05). In addition, levels of TryR protein were also markedly elevated in 3 out of 4 MAR isolates (range: 2.2-4.1 fold). This was accompanied by overexpressed TryR activity (mean level of 46.83±2.43 for extracts of MAR vs. 20.98±3.02 for MAS strains) (P< 0.05). Elevated levels of TryP, active enzyme in peroxide detoxification, were observed in MAR parasites resulting in an increased metabolism of H2O2. TryR activity was overexpressed on average in extracts of MAR strains, but not in all isolates. Enhanced anti-oxidant defenses through thiol metabolism may play a significant role in clinical resistance of ACL patients to Glucantime.

Upregulation of abeM, amvA, and qacEΔ1 efflux pump genes associated with resistance of Acinetobacter baumannii strains to disinfectants.

BACKGROUND AND AIMS: Acinetobacter baumannii is among the most concerning cause of nosocomial infections due to its high level of antibiotic resistance and high mortality. The aim of this study was to determine the role of efflux pumps in resistance of A. baumannii strains to three disinfectants, including MICROZED ID-MAX, NANOSIL D2, and OPIDEX OPA. METHODS: Twenty-eight environmental and clinical isolates of A. baumannii were collected from selected hospitals of central Iran. The minimum inhibitory concentrations of the disinfectants were determined and real time reverse transcriptase-PCR was performed to investigate the expression level of qacEΔ1, amvA, abeM, and adeB efflux pump genes. RESULTS: Considering both clinical and environmental isolates, there was a significant difference in the mean expression level of qacEΔ1 gene between susceptible and resistant strains to MICROZED ID-MAX disinfectant, of amvA and abeM genes between susceptible and resistant strains to NANOSIL D2 disinfectant and of abeM gene in susceptible and resistant strains to OPIDEX OPA disinfectant (all P Ë‚ .05). The expression levels of abeM and amvA genes were higher in the environmental isolates that were resistant to NANOSIL D2 disinfectant compared to those that were susceptible (P Ë‚ .05). CONCLUSIONS: This study provided evidence for the role of abeM and amvA genes in the resistance of environmental isolates to disinfectants, particularly hydrogen peroxide derivatives.

Differential inflammatory responses associated with Leishmania major and L tropica in culture.

BACKGROUND: Anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica and zoonotic CL (ZCL) due to L major have different clinical and epidemiological features. OBJECTIVES: To determine whether pro-inflammatory cytokines are involved in diverse pathogenicity of Leishmania species causing CL. PATIENTS/METHODS: The capacity of L major/L tropica to modulate expression of IL-1ß, IL-8 (CXCL8), IFN-γ, TNF-α and MCP-1 (CCL2) in peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) was evaluated by real-time RT-PCR technique. RESULTS: PBMCs from both ZCL and ACL cases expressed significantly higher IFN-γ (P < .001) and TNF-α (P < .05) compared with healthy controls (HC). PBMCs from ACL patients expressed significantly higher IL-1ß and IL-8 compared with ZCL patients and HC when stimulated with live L major or L tropica promastigotes (P < .001). After 4 and 10 hours, L major-infected MDMs expressed significantly higher IFN-γ (P < .05), and after 10 hours, L tropica-infected MDMs expressed significantly higher IL-1ß, IFN-γ and IL-8 compared with noninfected cells (P < .05). CONCLUSIONS: This study shows differential parasite-mediated stimulations of the inflammatory response with L major vs L tropica ex vivo. Pro-inflammatory cytokines particularly IL-8 (CXCL8) and IL-1ß might contribute in diverse clinical features of CL such as longer duration of lesion persistence in ACL patients.

Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis.

BACKGROUND: The characterization of Leishmania species is important for clinical management of the diseases and the epidemiological control of the parasite distribution. Most of the published polymerase chain reaction (PCR) amplification methods lack the ability to identify all species complexes, have low performance and usually need post-PCR procedures. There is a need for improving the diagnosis of CL by development of an accurate affordable PCR method to identify all Leishmania species in clinical specimens. METHODS: We designed an optimized a PCR amplifying the internal transcribed spacer 2 sequence of the ribosomal RNA gene (ITS2) aligned from different strains of CL-causing Leishmania species in the Old World. The performance of the method was evaluated on lesion samples from several CL suspected patients and the limit of detection (LOD) was determined on DNA of promastigotes from reference strains. RESULTS: The universal PCR enabled simultaneous discrimination of L. major, L. tropica and L. infantum using one primer pair in one reaction. Stained smear microscopy and Novy-MacNeal-Nicolle (NNN) medium culture alone detected 77.27% (17/22) and 72.73% (16/22) of the positive CL samples, respectively. The PCR assay showed 100% sensitivity (22/22) (95% CI: 84.56-100%) and 100% specificity (3/3) (95% CI: 29.24-100%) for species identification on isolates from lesion scraping/exudate and 100% sensitivity (13/13) (95% CI: 75.29-100%) and 100% specificity (11/11) (95% CI: 71.51-100%) for species identification on biopsy samples of CL patients, while being capable to successfully amplify as little as 0.01-0.1 pg of Leishmania DNA from cultured promastigotes. CONCLUSIONS: We present a validated easy-to-use affordable universal PCR assay to identify the most common Old World Leishmania species causing CL. This PCR assay could be used as a sensitive/specific technique to diagnose CL-causing Leishmania species in clinical samples with high accuracy.