In-vitro antifungal susceptibility testing of lanoconazole and luliconazole against Aspergillus flavus as an important agent of invasive aspergillosis.

INTRODUCTION: The incidence of Aspergillus infections has recently increased remarkably in certain tropical and sub-tropical countries, with Aspergillus flavus being identified as the leading cause of infections after A. fumigatus. Lanoconazole (LAN) and luliconazole (LUL) are currently approved for topical treatment of cutaneous fungal infections. We aimed the in-vitro antifungal susceptibility testing of two imidazole, LAN and LUL against A. flavus. METHODS: One hundred and eighty-seven clinical and environmental A. flavus were tested originating from different climate zones of Iran between 2008 and 2015. The identification of all isolates was confirmed by using PCR-sequencing of ß-tubuline ribosomal DNA gene. In-vitro antifungal susceptibility test was performed using CLSI guidelines against LAN, LUL, itraconazole (ITC), voriconazole (VRC), posaconazole (POS), Isavuconazole (ISA), amphotericin B (AMB), 5-flucytosine (5FC), caspofungin (CAS) and anidulafungin (AFG). The minimum inhibitory concentration (MIC) and minimum effect concentration (MEC) values were evaluated according to CLSI M38-A2 guidelines. RESULTS: The geometric mean MICs for tested antifungals, in increasing order, were: 0.009 µg/mL for LUL (ranging from 0.004 to 0.062), 0.02 µg/mL for LAN (ranging from 0.004 to 0.125), POS (0.10), ISA (0.16), ITC (0.24), VRC (0.27), AMB (1.8) and 5FC (63.06) µg/mL. The mean value of MECs for AFG and CAS were 0.06 and 0.07, respectively. CONCLUSION: Overall, LUL and LAN showed the lowest MIC against all isolates of A. flavus. Further studies are required to evaluate the in-vivo efficacy of these agents, and the possibility of using these agents in systemic infections.

Effect of involved Aspergillus species on galactomannan in bronchoalveolar lavage of patients with invasive aspergillosis.

PURPOSE: The detection of galactomannan (GM) in bronchoalveolar lavage (BAL) fluid is an important surrogate marker for the early diagnosis and therapeutic monitoring of invasive aspergillosis (IA), regardless of the involved species of Aspergillus. Here, we utilized the Platelia Aspergillus GM enzyme immunoassay (Bio-Rad) to evaluate the GM index in BAL fluid samples from patients with proven, probable or putative IA due to Aspergillusflavus versus Aspergillusfumigatus. METHODOLOGY: In a prospective study between 2009 and 2015, 116 BAL samples were collected from suspected IA patients referred to two university hospitals in Tehran, Iran. KEY FINDINGS: According to European Organization for Research and Treatment of Cancer and Mycoses Study Group and Blot criteria, 35 patients were classified as IA patients, of which 33 cases tested positive for GM above 0.5 and, among these patients, 22 had a GM index ≥1. Twenty-eight were culture positive for A. flavus and seven for A. fumigatus. The GM index for A. flavus cases was between 0.5-6.5 and those of A. fumigatus ranged from 1 to 6.5. The sensitivity and specificity of a GM index ≥0.5 in cases with A. flavus were 86 and 88 % and for A. fumigatus patients were 100 and 73 %, respectively. CONCLUSION: Overall, the mean GM index in patients with A. fumigatus (3.1) was significantly higher than those of A. flavus (1.6; P-value=0.031) and the sensitivity of GM lower for A. flavus when compared to A. fumigatus. This finding has implications for diagnosis in hospitals and countries with a high proportion of A. flavus infections.

Genetic Diversity and In Vitro Antifungal Susceptibility of 200 Clinical and Environmental Aspergillus flavus Isolates.

Aspergillus flavus has been frequently reported as the leading cause of invasive aspergillosis in certain tropical and subtropical countries. Two hundred A. flavus strains originating from clinical and environmental sources and collected between 2008 and 2015 were phylogenetically identified at the species level by analyzing partial ß-tubulin and calmodulin genes. In vitro antifungal susceptibility testing was performed against antifungals using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. In addition, genotyping was performed using a short-tandem-repeat (STR) assay of a panel of six microsatellite markers (A. flavus 2A, 2B, 2C, 3A, 3B, and 3C), in order to determine the genetic variation and the potential relationship between clinical and environmental isolates. The geometric means of the minimum inhibitory concentrations/minimum effective concentrations (MICs/MECs) of the antifungals across all isolates were (in increasing order): posaconazole, 0.13 mg/liter; anidulafungin, 0.16 mg/liter; itraconazole, 0.29 mg/liter; caspofungin, 0.42 mg/liter; voriconazole, 0.64 mg/liter; isavuconazole, 1.10 mg/liter; amphotericin B, 3.35 mg/liter; and flucytosine, 62.97 mg/liter. All of the clinical isolates were genetically different. However, an identical microsatellite genotype was found between a clinical isolate and two environmental strains. In conclusion, posaconazole and anidulafungin showed the greatest in vitro activity among systemic azoles and echinocandins, respectively. However, the majority of the A. flavus isolates showed reduced susceptibility to amphotericin B. Antifungal susceptibility of A. flavus was not linked with the clinical or environmental source of isolation. Microsatellite genotyping may suggest an association between clinical and environmental strains, although this requires further investigation.

Nocardia isolation from clinical samples with the paraffin baiting technique.

BACKGROUND: The genus Nocardia is a cause of infection in the lungs, skin, brain, cerebrospinal fluid, eyes, joints and kidneys. Nocardia isolation from polymicrobial specimens is difficult due to its slow growth. Several methods have been reported for Nocardia isolation from clinical samples. In the current study, we used three methods: paraffin baiting technique, paraffin agar, and conventional media for Nocardia isolation from various clinical specimens from Iranian patients. METHODS: In this study, we examined 517 samples from various clinical specimens such as: sputum of patients with suspected tuberculosis, bronchoalveolar lavage, sputum of patients with cystic fibrosis, tracheal aspirate, cutaneous and subcutaneous abscesses, cerebrospinal fluid, dental abscess, mycetoma, wound, bone marrow biopsy, and gastric lavage. All collected specimens were cultured on carbon-free broth tubes (paraffin baiting technique), paraffin agar, Sabouraud dextrose agar, and Sabouraud dextrose agar with cycloheximide and were incubated at 35°C for one month. RESULTS: Seven Nocardia spp. were isolated with paraffin baiting technique, compared with 5 positive results with the paraffin agar technique and 3 positive results with Sabouraud dextrose agar with and without cycloheximide. The prevalence of nocardial infections in our specimens was 5.28%. CONCLUSION: In the present study, the use of the paraffin baiting technique appeared to be more effective than other methods for Nocardia isolation from various clinical specimens.

Effects of crystal pixel size and collimator geometry on the performance of a pixelated crystal gamma-camera using Monte Carlo simulation.

Dedicated γ-cameras based on pixelated scintillators have long been used for breast tumor imaging. Intercrystal scattering (ICS) increases the background counting rate and degrades the image quality when small crystal pixels are used. Because of the small size of applied collimators, scattered radiation and septal penetration are high, and therefore collimator characteristics must be carefully considered. In our study, we investigated the influence of ICS events on position-detection accuracy (PDA) for pixelated crystals and the effects of different geometries of hexagonal-hole collimators on the performance of these cameras, using Monte Carlo simulation to optimize camera design. The arrays of thallium-doped cesium iodide detectors with different pixel dimensions that had been exposed to 140-keV photons of isotropic point source, 50 mm from the collimator surface, were simulated. Hexagonal-hole collimators were 10.5, 15, and 21 mm long. The septal thickness varied from 0.1 to 0.5 mm, with 3 different hole diameters. The results confirmed that by increasing the crystal pixel size, ICS was decreased and change of detection efficiency was negligible, but PDA, contrast-to-noise ratio, and spatial resolution (full width at half maximum) were increased. Our experiences confirmed that 2 × 2 mm was an optimum crystal pixel size, especially for a lower ICS fraction and an appropriate full width at half maximum. Because collimators are the limiting factor for spatial resolution and sensitivity, careful collimator design is of great importance.