A. A. M. A. Baqui

Evaluation of a Reformulated CHROMagar Candida

ABSTRACT CHROMagar Candida is a differential culture medium for the isolation and presumptive identification of clinically important yeasts. Recently the medium was reformulated by Becton Dickinson. This study was designed to evaluate the performance of the new formula of CHROMagar against the original CHROMagar Candida for recovery, growth, and colony color with stock cultures and with direct plating of clinical specimens. A total of 90 stock yeast isolates representing nine yeast species, including Candida dubliniensis, as well as 522 clinical specimens were included in this study. No major differences were noted in growth rate or colony size between the two media for most of the species. However, all 10 Candida albicans isolates evaluated consistently gave a lighter shade of green on the new CHROMagar formulation. In contrast, all 26 C. dubliniensis isolates gave the same typical dark green color on both media. A total of 173 of the 522 clinical specimens were positive for yeast, with eight yeast species recovered. The recovery rates for each species were equivalent on both media, with no consistent species-associated differences in colony size or color. Although both media were comparable in performance, the lighter green colonies ofC. albicans isolates on the new CHROMagar made it easier to differentiate between C. albicans and C. dubliniensis isolates. In conclusion, the newly formulated Becton Dickinson CHROMagar Candida medium is as equally suited as a differential medium for the presumptive identification of yeast species and for the detection of multiple yeast species in clinical specimens as the original CHROMagar Candida medium.

Enhanced Interleukin-1β, Interleukin-6 and Tumor Necrosis Factor-α Production by LPS Stimulated Human Monocytes Isolated from HIV + Patients

Abstract Periodontal disease and tooth loss is a common finding among advanced HIV+ patients. In addition to local oral lipopolysaccharide (LPS) stimulation, systemic up-regulation of monocyte pro-inflammatory cytokine secretion may also be involved in the pathogenesis of HIV disease. A study was undertaken to investigate IL-1β, IL-6 and TNF-α production by resting and LPS stimulated monocytes isolated from HIV + patients and also to investigate the relationship of the patients HIV viral load status to the cytokine production. Whole blood samples in EDTA were collected from 39 HIV-1 infected patients and 20 age and sex matched uninfected controls. Plasma was separated by centrifugation. Viral load was determined using a quantitative RT-PCR. Monocytes were isolated by Ficoll-hypaque gradient separation followed by overnight plastic adherence. Cultured monocytes (1 × 106ml) were stimulated with LPS (1 μg/ml) of either P. gingivalis or F. nucleatum for 2, 8, 24 and 48 h and supernatant fluids were collected. IL-1β, IL-6, and TNF-α levels in supernatant fluids were estimated by ELISA. Increased overall production of IL-1β, IL-6 and TNF-α by LPS stimulated monocytes isolated from HIV-1 infected patients was observed when compared to HIV-1 uninfected controls. LPS stimulated monocytes from HIV-1 infected patients with high viral load (HVL) produced significant (p<0.05) elevations in these pro-inflammatory cytokines when compared to HIV-1 uninfected controls. Both LPS of P. gingivalis and F. nucleatum produced a comparable cytokine production by monocytes after 8 h of stimulation. These data suggest that enhanced IL-1β, IL-6 and TNF-α is produced by monocytes/macrophages isolated from HVL HIV + patients and may be involved in the overall pathogenesis of HIV-1 infection.

The effects of HIV viral load on the phagocytic activity of monocytes activated with lipopolysaccharide from oral microorganisms.

A study was undertaken to determine whether viral load status in HIV+ patients has any potential effect on monocyte phagocytic function both before and after challenge of the monocytes with lipopolysaccharide (LPS) isolated from oral microorganisms. LPS of two putative periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) was prepared. Whole blood samples in EDTA were collected from 30 HIV+ patients presenting for dental care at the University of Maryland. Control samples were prepared from appropriate uninfected individuals. Viral load was determined using quantitative RT-PCR (Amplicor, Roche Diagnostics). Phagocytic function was determined using FITC labeled Saccharomyces species in resting isolated monocytes and in cells after 24 h stimulation with 1 microgram/ml of LPS of P. gingivalis or F. nucleatum. Immunohistochemical staining was performed for complement receptor CR-1 (CD-35) on phagocyte cells. In HIV+ patients with high viral load (> 10,000 copies/ml), 13.5% of isolated resting monocytes demonstrated phagocytic activity, while 23% of the resting control monocytes from non-infected individuals showed phagocytic function. When the monocytes were stimulated with 1 microgram/ml of LPS of F. nucleatum, phagocytic activity was observed in 18.5% of monocytes in patients with high viral load, 33.5% with moderate viral load (400-10,00 copies/ml) and 51% with low viral load (<400 copies/ml), while 62% of the control monocytes demonstrated phagocytic activity. Stimulation of monocytes with LPS of P. gingivalis showed similar results. Complement receptor CD-35 showed a 50% decrease in expression in HIV+ patients with high viral load. A progressive decrease in monocyte/macrophage phagocytic function and CD-35 expression with and without oral LPS activation occurs after HIV infection and this trend appears to be accentuated in patients with high viral load. This relationship may contribute to increased susceptibility to oral opportunistic infections in advanced HIV+ patients.

Functional changes in THP-1 human monocytic cells after stimulation with lipopolysaccharide of oral microorganisms and granulocyte macrophage colony stimulating factor

A human THP-1 monocyte cell line culture system has been utilized to observe the effect of granulocyte macrophage colony stimulating factor (GM-CSF) supplementation with lipopolysaccharide (LPS) of oral microorganisms to stimulate monocyte/macrophage functional activity. LPS of oral microorganisms, Fusobacterium nucleatum and Porphyromonas gingivalis was produced by phenol-water extraction and characterized. The phagocytosis assay was performed using F1TC labeled Saccharomyces yeast particles. Phagocytic functional activity was observed in 10-11% of resting THP-1 cells. Treatment of THP-1 cells with LPS of F. nucleatum or P. gingivalis increased the phagocytic activity of THP-1 cells 2-3 fold. GM-CSF significantly increased phagocytosis either alone or when supplemented with LPS of F. nucleatum or P. gingivalis. A chemotaxis assay was performed using a 48 well chemotaxis chamber. Chemotactic functional activity of THP-1 cells was increased 2-fold after 4 days of treatment with GM-CSF. Stimulation of THP-1 cells with LPS of F. nucleatum or P. gingivalis significantly reduced the chemotactic activity indicating the maturation towards a fixed macrophage. There were functional variations (chemotaxis and phagocytosis) in THP-1 cells in response to LPS of oral microorganisms following stimulation with GM-CSF.