Fluconazole susceptibility testing of Cryptococcus neoformans: comparison of two broth microdilution methods and clinical correlates among isolates from Ugandan AIDS patients

We compared the yeast nitrogen base (YNB) broth microdilution method with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A microdilution reference method for measuring the in vitro susceptibility of Cryptococcus neoformans isolates to fluconazole. A total of 149 isolates of C. neoformans var. neoformans from Ugandan AIDS patients was tested by both methods. An overall agreement of 88% between the two microdilution methods was observed. All isolates grew well in both RPMI 1640 and YNB media, and MICs could be read after 48 h of incubation by both methods. The range of fluconazole MICs obtained with the YNB method was broader than that obtained with the NCCLS method. The extended range of MICs provided by the YNB method may be of clinical value, as it appears that the clinical outcome may be better among patients infected with strains inhibited by lower concentrations of fluconazole as determined by the YNB method. The YNB method appears to be a viable option for testing C. neoformans against fluconazole.     

In vitro growth-inhibitory activity of pneumocandins L-733,560 and L-743,872 against putatively amphotericin B- and fluconazole-resistant Candida isolates: influence of assay conditions

L-733,560 and L-743,872 are water-soluble pneumocandins with potent antifungal activity. By beginning with the NCCLS M27-T method and varying test format (microdilution vs macrodilution), time of reading (24 h vs 48 h), and test medium (RPMI 1640 vs Antibiotic Medium 3), we have demonstrated that the MICs for these compounds can be altered significantly: the microdilution format, reading after 24 h and use of Antibiotic Medium 3 all reduced the measured MIC. No cross-resistance was demonstrated with either fluconazole or amphotericin B.     

Detection of resistance to amphotericin B among Cryptococcus neoformans clinical isolates: performances of three different media assessed by using E-test and National Committee for Clinical Laboratory Standards M27-A methodologies

Although reliable detection of resistance in vitro is critical to the overall performance of any susceptibility testing method, the recently released National Committee for Clinical Laboratory Standards M27-A methodology for susceptibility testing of yeasts discriminates poorly between resistant and susceptible isolates of Candida spp. We have previously shown that both substitution of antibiotic medium 3 for RPMI 1640 medium in the microdilution variant of the M27-A method and use of the E-test agar diffusion methodology permit detection of amphotericin B-resistant Candida isolates. To determine the relevance of these observations to Cryptococcus neoformans, we have evaluated the performances of both the M27-A and the E-test methodologies with this yeast using three different media (RPMI 1640 medium, antibiotic medium 3, and yeast nitrogen base). As with Candida, we found that only antibiotic medium 3 permitted consistent detection of resistant isolates when testing was performed in broth by the M27-A method. When testing was performed by the E-test agar diffusion method, both RPMI 1640 medium and antibiotic medium 3 agar permitted ready detection of the resistant isolates. Reading of the results after 48 h of incubation was required for testing in broth by the M27-A method, while the MIC could be determined after either 48 or 72 h when the agar diffusion method was used.     

Iron repressible outer membrane proteins of Moraxella bovis and demonstration of siderophore-like activity

Moraxella bovis (strain Epp 63), grown in RPMI 1640 medium supplemented with desferrioxamine mesylate (0.05 mg/ml) resulted in cell free culture supernatants with an increased chromeazurol-S response indicating the presence of high affinity iron binding ligand(s). Supernatants of cultures where growth occurred in tryptic soy broth, RPMI 1640, or RPMI 1640-desferrioxamine supplemented with ferrous sulfate (10 micrograms/ml) were negative on the chromeazurol-S test. Growth of M. bovis in RPMI 1640 or RPMI 1640-desferrioxamine medium induced the expression of previously unrecognized outer membrane proteins whose expression was repressed when the medium was supplemented with iron and which were not produced when growth occurred in tryptic soy broth.     

In-vitro susceptibility pattern of Candida lusitaniae and evaluation of the Etest method

The antifungal susceptibility of 35 Candida lusitaniae isolates was determined in vitro by the National Committee for Clinical Laboratory Standards (NCCLS) M27-P macrodilution methodology. All the isolates were susceptible to ketoconazole, itraconazole and fluconazole. Of the 35 isolates, eight (23%) were resistant to flucytosine. For amphotericin B, M27-P yielded a narrow range of MICs (0.06-0.5 mg/L). We therefore investigated the activity of this drug by reading MICs at 72 h and by using alternative media, namely casitone complex medium (CCM) and antibiotic medium 3 (M-3). Reading at 72 h did not generate reproducible results. CCM and M-3 provided better discrimination between the isolates but did not change the rank order of the MICs. We thus concluded that all the isolates were susceptible to amphotericin B. We also conducted an evaluation with the Etest method according to the manufacturer's instructions with RPMI 1640 agar, CCM and the alternative semi-synthetic medium (SSM). For RPMI 1640, agreements +/-2 dilutions were 58% for amphotericin B, 92% for flucytosine, 57% for ketoconazole, 92% for fluconazole and 74% for itraconazole. CCM did not improve the agreement rates between the two methods but it led to better growth of all the isolates. The suitability of SSM was pointed out with itraconazole. The poor agreement rates for amphotericin B and ketoconazole call for further evaluation of the Etest method to assess several drug-organism combinations.     

Comparison of a photometric method with standardized methods of antifungal susceptibility testing of yeasts

We determined the fluconazole MICs for 101 clinical isolates of Candida and Cryptococcus neoformans using the macro- and microdilution methods recommended by the National Committee for Clinical Laboratory Standards. We compared the MICs obtained by these methods with those obtained by a photometric assay that quantified the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) by viable fungi. The MIC determined by this method was defined as the highest fluconazole concentration associated with the first precipitous drop in optical density. For Candida, both the MTT and the microdilution methods demonstrated excellent agreement with the standard macrodilution method. The MTT method, however, generated MICs at 24 h that were comparable to those generated by the standard macrodilution method, whereas the microdilution method required 48 h. For C. neoformans, the levels of agreement between the MICs determined by the MTT and microdilution methods after 48 h and those determined by the standard 72-h macrodilution method were 94% (29 of 31) and 94% (29 of 31), respectively. The MTT method therefore provided results comparable to those of currently recommended methods and had the advantages of a more rapid turnaround time and potential adaptability to use as an automated system. Furthermore, the MICs determined by the MTT method were determined photometrically, thereby eliminating reader bias.     

Age-related changes in albumin-excluded volume fraction

A broth microdilution antifungal susceptibility test with a pH indicator (bromocresol purple) in a synthetic medium was evaluated. This method measures cellular activity instead of simply a change in biomass. The variations of pH caused by fungal activity were measured by changes in optical densities at 450 nm. The MICs50 obtained were compared with the MICs found by the classical broth macrodilution procedure. In most cases broth micro- and macrodilution MICs were in agreement for amphotericin B, fluconazole, flucytosine and nystatin.     

Delicate and crucial pH dependence in permeabilization of cultured mammalian cells by vortex-stirring with a high molecular weight polyacrylic acid

We previously reported that cultured mammalian cells are effectively permeabilized by vortex-stirring the cells with a high molecular weight polyacrylic acid. The present study revealed that the efficiency of permeabilization of a non-permeant dye, Lucifer Yellow (LY), was sensitively affected by the pH of the medium. When the cells are vortex-stirred in RPMI 1640 culture medium, the pH of the medium should be adjusted with HEPES and NaOH, instead of NaHCO3, to a pH higher than 7.6 for successful permeabilization; the higher the pH, the better the result obtained. Internalization of LY was near the background level at a pH below 7.4. When phosphate-buffered saline was substituted for RPMI 1640 medium, the optimal pH range was slightly shifted to a more acidic region. This requirement for the pH of the medium is indispensable as a supplement to the standard permeabilization procedure tentatively proposed in the preceding report.     

Influence of the culture medium on the expression of surface polypeptides of Yersinia enterocolitica

Three polypeptides (200, 46, and 25 kDal) encoded by the virulence plasmid were detected by SDS-PAGE in the outer membrane of Yersinia enterocolitica 09 grown at 37 degrees C in brain-heart infusion medium. Bacteria grown at the same temperature in the tissue culture medium RPMI 1640 expressed five additional polypeptides (170, 135, 118, 100, and 98 kDal), but the 25-kDal band was not seen. The protein profile in RPMI 1640 resembles the expression pattern displayed by yersiniae when grown in vivo. The immunoblot of total membrane proteins of bacteria grown in brain-heart infusion medium revealed eight plasmid-encoded polypeptides, four of which were also in the outer membrane preparations, including a 28-kDal polypeptide. These peptides do not coincide with known plasmid-encoded outer membrane proteins.     

Comparative evaluation of National Committee for Clinical Laboratory Standards broth macrodilution and agar dilution screening methods for testing fluconazole susceptibility of Cryptococcus neoformans

A simple screening method for fluconazole susceptibility of Cryptococcus neoformans using 2% dextrose Sabouraud dextrose agar (SabDex) with fluconazole was compared to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method. By this method, fluconazole-susceptible C. neoformans isolates are significantly smaller on medium with fluconazole than on fluconazole-free medium. Isolates with decreased susceptibility have normal-size colonies on medium containing fluconazole. The 48-h NCCLS broth macrodilution MICs (NCCLS MICs) for isolates with normal-size colonies on 8- or 16-microg/ml fluconazole plates were predicted to be > or =8 or > or =16 microg/ml, respectively. On medium with 16 microg of fluconazole per ml, all strains (84 of 84) for which the NCCLS MICs were <16 microg/ml were correctly predicted, as were all isolates (7 of 7) for which the MICs were > or =16 microg/ml. Agar dilution appears to be an effective screening method for fluconazole resistance in C. neoformans.     

Evaluation of endpoints for antifungal susceptibility determinations with LY303366

We have previously reported poor correlation between the in vitro fungicidal activity of LY303366 and MIC results in RPMI medium based upon the manufacturer's suggested susceptibility endpoint, lack of visual growth. Additionally, we have noted a significant trailing effect with LY303366 when MICs are determined in RPMI medium. These observations have led us to evaluate an alternative susceptibility endpoint for LY303366, an 80% reduction in growth compared with control (similar to that utilized for azoles). Two isolates each of Candida albicans, Candida glabrata, and Candida tropicalis were selected for testing. MICs were determined for LY303366 in RPMI 1640 medium buffered with morpholinepropanesulfonic acid. MICs were determined with suggested (MIC100) and experimental (MIC80) endpoints. The minimal fungicidal concentration (MFC) of LY303366 for each isolate was also determined. Time-kill curves were determined in RPMI medium with each isolate at concentrations of LY303366 ranging from 0.125 to 16x MIC80 to assess the correlation between MIC80 and fungicidal activity. Lastly, fungi exposed to LY303366 were examined via scanning electron microscope (SEM) for evidence of drug-induced ultrastructure change. MIC80s for test isolates ranged from 0.015 to 0.12 microgram/ml and were consistently three to five wells less than MIC100s. Good correlation was observed between fungicidal activity, as assessed by kill curves, and the MIC80. SEM data revealed significant ultrastructure changes induced by LY303366 even at sub-MIC80s. Based on our results demonstrating better correlation between MIC80 and fungicidal activity, i.e., time-kill curves and MFCs, we suggest that 80% reduction in visible growth be utilized as the endpoint for susceptibility determinations with LY303366 in RPMI medium.     

Stability of artemisinin in aqueous environments: impact on its cytotoxic action to Ehrlich ascites tumour cells

We have recently shown artemisinin to be cytotoxic against Ehrlich ascites tumour cells. The aim of this study was to investigate the stability of this compound in the aqueous environment of the in-vitro Ehrlich ascites tumour cell system (RPMI 1640 cell culture medium supplemented with 10% foetal bovine serum (RPMI/FBS) with reference to its cytotoxic action. Literature data show that artemisinin can react with Fe2+ yielding reactive intermediates leaving artemisinin G as a major end-product. The current study showed that only excess addition of Fe2+ to artemisinin in distilled water, phosphate-buffered saline (PBS) and RPMI/FBS and incubation for 24 h led to degradation of artemisinin and yielded artemisinin G. If Fe2+ was not added results from HPLC analysis were indicative of complete recovery of artemisinin from distilled water and RPMI/FBS, with or without cells, at 37 degrees C for at least 24 h. In addition, incubation of artemisinin in RPMI/FBS with or without cells at 37 degrees C for 24 h before cytotoxicity assay did not change its cytotoxic action. On the basis of these results, we suggest that cytotoxicity to tumour cells was caused by unchanged artemisinin. This is not so for the antimalarial activity of artemisinin and derivatives, for which the presence of a pool of (haem) Fe2+ is a prerequisite resulting in free radicals or electrophilic intermediates or both.     

Fragile sites on human chromosomes: demonstration of their dependence on the type of tissue culture medium

The observation of heritable fragile sites on human chromosomes prepared for lymphocyte cultures has been shown to depend on the type of tissue culture medium in which the lymphocytes are grown. The sites are observed at a much greater frequency when medium 199 is used than when RPMI 1640, Ham's F10, Eagle's (basal), and CMRL 1969 are used. One site on the X chromosome is of clinical significance in that it is a marker for X-linked mental retardation.     

Expression of macrophage markers by a population of T cells obtained from synovial fluid of a subgroup of patients with juvenile rheumatoid arthritis

OBJECTIVE: To characterize distinctive lymphoid cell populations in the synovial fluid (SF) of patients with juvenile rheumatoid arthritis (JRA) that have the specific ability to display monocytic markers when cultured in vitro. METHODS: Mononuclear cells obtained from SF of patients with JRA and depleted of adherent macrophages were cultured in vitro in RPMI 1640 medium supplemented with only fetal calf serum (FCS). Phenotypic evaluation of these cells was by flow cytometry and immunohistochemical analysis was by specific fluorochrome labeled antibodies. RESULTS: T cells from a JRA subgroup displayed some typical macrophage attributes, i.e., abundant cytoplasm, adherence to plastic, and phagocytosis of latex beads when cultured in vitro. These cells have the ability to survive in culture for several weeks in RPMI 1640 medium containing only 10% FCS. The macrophage-like T cells rosetted with sheep red blood cells and proliferated when stimulated with phytohemagglutinin or anti-CD3, indicating functional T cell responses. CONCLUSION: Our data indicate that a population of T cells obtained from the SF of a subgroup of patients with JRA exhibited characteristics of macrophages, yet retained their CD3 and T cell receptor expression. Whether this promiscuous behavior is caused by malignant transformation of lymphoid precursor cells or is induced by the concerted effect of a myriad of cytokines and growth factors present in the SF remains unknown. The presence of these cells in the SF of 2 patients with JRA with different onset types raises the question of their function and significance in an autoimmune disorder such as JRA.     

Pregnancies from subzonal insemination of unfertilized oocytes after conventional IVF

Babesia caballi infected erythrocytes were collected from the blood of an experimentally infected horse and could be continuously cultivated in vitro with parasitemia ranging from 2-4% in RPMI 1640 medium supplemented with 2 mM L-glutamine, 20 mM HEPES and 40% adult horse serum in a low oxygen atmosphere (2% O2, 5% CO2 and 93% N2). All attempts to increase parasitemia failed using other culture media, serum concentrations and culture vessels. However, parasite growth was enhanced by transfer of cultures from a low oxygen to 5% CO2 in air, with parasitemia ranging from 8-10%.     

In vitro cultivation of Trypanosoma acomys: production of insect stages and bloodstream forms

When Trypanosoma acomys bloodstream forms were cultivated at 37 degrees C in Schneider's Drosophila medium supplemented with 20% (v/v) heat-inactivated foetal calf serum (FCS), with Microtus agrestis embryonic fibroblasts in RPMI 1640 medium supplemented with 20% FCS or in Baltz's medium supplemented with 10% FCS, the parasites transformed and largely remained as epimastigotes. Epimastigotes were also usually the commonest stage observed when the parasites were co-cultivated with a mosquito cell line at 27 degrees C. However, if these cultures were initiated with the supernatant suspensions from fibroblast cultures that had been cryopreserved, trypomastigotes, including bloodstream-like forms, were the predominant stage for the first 4 days of culture. It is suggested that the glycerol supplement or the temperature changes stimulated this unusual morphogenesis. At 27 degrees C, T. acomys was incapable of multiplying and died when cultured in fresh Schneider's Drosophila medium supplemented with 20% FCS, but co-cultivation with the mosquito cell lines or cultivation in cell-free supernatants from 1-week-old mosquito cell cultures was successful at this temperature; most of the parasites multiplied as epimastigotes.     

Tranilast inhibits the growth of rat mesangial cells

We investigated the effects of tranilast on the growth of cultured rat mesangial cells. The number of mesangial cells increased fivefold during a 5-day incubation in RPMI 1640 with 20% fetal bovine serum. The number of cells was significantly lower in the presence of tranilast than in its abscence. Tranilast (0 approximately 500 microM) inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis of rat mesangial cells cultured in RPMI 1640 medium containing 0.5% fetal bovine serum in a dose-dependent manner. The inhibition of DNA synthesis by tranilast was not affected by the presence of indomethacin (1 microg/ml) or N(G)-monomethyl-L-arginine (0.5 mM). Tranilast did not stimulate nitrite oxide synthesis in PDGF-stimulated cells. Mitogen-activated protein kinase activity in mesangial cells was significantly increased by exposure to PDGF, while the effect was significantly suppressed in the presence of tranilast. The present study revealed that tranilast inhibits the growth of rat mesangial cells, independently of nitric oxide or prostacycline synthesis.     

Temperature effects on Legionella pneumophila killing by and multiplication in phagocytes of guinea pigs

We examined the effects of temperature on the interaction between Legionella pneumophila and phagocytes of guinea pigs. The body temperatures of guinea pigs infected with a sublethal dose (1.2 x 10(4) CFU) or a lethal dose (1.0 x 10(5) CFU) of L. pneumophila elevated from 38.4 +/- 0.15 C to 40.2 +/- 0.42 C or 40.3 +/- 0.62 C, respectively. The intracellular bacterial killing by and bacterial proliferation in the phagocytes were examined at 33, 37, 40, and 42 C, using in vitro culture systems of peritoneal macrophages or polymorphonuclear leukocytes (PMN) of guinea pigs. In all the macrophages incubated at different temperatures, significant intracellular bacterial killings were observed at 4 hr after in vitro phagocytosis. After 24 hr of incubation, there was about a 100-fold increase of CFU and the number reached a maximum after 48 hr of incubation in the macrophages incubated at 42 C as well as 37 and 40 C, suggesting that macrophages support the intracellular bacterial growth in hyperthermia. In the PMN, L. pneumophila CFU 4 hr or 12 hr after the infection were significantly lower at 42 C than those at 37 C (P < 0.05), indicating that the bactericidal capacity of PMN was enhanced at 42 C compared to 37 C. However, in all the PMN incubated at different temperatures, there were about 10-fold increases of CFU 24 hr after the infection, suggesting that PMN as well as macrophages support intracellular bacterial growth in hyperthermia. The extracellular bacterial growth was examined at 33, 37, 40, and 42 C in buffered yeast extract (BYE) broth or RPMI 1640 medium containing 50% guinea pig serum as a permissive or non-permissive liquid medium for the bacterial growth, respectively. Inhibition of bacterial growth in BYE broth at 42 C, and a decrease of CFU in RPMI 1640 medium containing 50% guinea pig serum at 42 C were observed. In conclusion, hyperthermia may be beneficial by restricting extracellular bacterial survival, but it exerts no beneficial effect on the restriction of intracellular bacterial growth in phagocytes, though PMN showed enhanced initial killing at 42 C. These results suggest that fever, or hyperthermia itself, may not largely contribute as a nonspecific host defense early in the course of legionellosis.     

Culture of endothelial cells as an in vitro model for experimental and clinical research]

Culture of endothelial cell is gotten from human umbilical cord by enzymatic digestion. For the growth of cells in culture, medial RPMI 1640 with 20% mixed human serum (NHS) or 20% fetal calf serum (FCS), endothelial cell growth factor (ECGF) and heparin have been used. Plastic, 0.1% gelatin and fibronectin have been used as a fundament. Immune identification of endothelial cells was culture is performed by monoclonal antibodies on vWF:Ag. Homogeneous cell line in culture might be used as in vitro model in both experimental and practice work.     

Morphological changes of clefts in Plasmodium-infected erythrocytes under adverse conditions

Blood infected with human or rodent malaria parasites, Plasmodium falciparum or Plasmodium berghei, was exposed to higher pH, higher PO2, and lower temperature than those used in standard cultivation conditions. Parasitized blood was incubated for 20, 25, and 30 min with RPMI 1640 medium, 10% (vol/vol) serum, pH 8.0, at 20 degrees C in the air, conditions which are ultimately lethal to the asexual stages of malarial parasites. Markedly dilated clefts were observed in the cytoplasm of the malaria-infected erythrocytes so treated. These clefts can take up colloidal gold particles and macromolecules such as Protein A, rhodamine-dextran, and lucifer yellow-dextran. Such dilated clefts were not seen in the cytoplasm of infected erythrocytes that were incubated under normal cultivation conditions before fixation. These had slender clefts of the usual sort that did not take up colloidal gold particles and macromolecules.