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Cryptococcus neoformans in tropical northern Australia: predominantly variant gattii with good outcomes.

BACKGROUND: Infection with Cryptococcus neoformans is common in the Northern Territory of Australia. Disease is life threatening and treatment is prolonged and often complicated by the need for surgery and difficulties with medical therapy. AIMS: To document incidence, demography, risk factors, clinical features and outcomes of infection and to determine differences between gattii and neoformans varieties. METHODS: Case records of all patients (n = 35) diagnosed with cryptococcal infection at the Royal Darwin Hospital between 1976 and 1992 were reviewed retrospectively. Current status of patients was ascertained. Variety identification of isolates was determined by growth in canavanine-glycine-bromthymol blue agar. RESULTS: Of the 35 patients, 23 had meningitis, ten had pneumonia, one had a dermal infection and one had fungaemia with no obvious focus. Twelve (52%) meningitis cases and two (20%) pneumonia cases had no predisposing disease. Thirteen (57%) meningitis cases had concomitant pulmonary cryptococcosis. Twenty-nine patients with Aboriginal and six were Caucasian, with a relative risk for Aboriginals compared with non-Aboriginals of 20.6 (95% CI 8.6-49.5). Arnhemland was the commonest location of infection, with an annual incidence in Aboriginals of 0.14/1000. Fourteen (78%) of 18 isolates tested were C. neoformans var. gattii. Management was characterised by the frequent need for adjunctive surgery and prolonged or repeat courses of systemic antifungal therapy. Despite this, long-term outcomes are encouraging with a mortality of 14% overall and 9% in meningitis patients. The river red gum (Eucalyptus camaldulensis) has a limited distribution in Arnhemland and ongoing studies are seeking alternative environmental sources of C. neoformans var. gattii.

Adolescent↗

Enhanced gastrointestinal absorption of drugs in rats pretreated with the synthetic immunomodulator, levamisole.

The effect of levamisole on drug absorption from the rat small intestine has been investigated by means of an in-situ recirculation technique. The absorption of salicylic acid, sulphanilamide and aminopyrine was significantly increased by the intraperitoneal administration of levamisole (2 mg) 1 day before the absorption studies, but there was no significant effect on absorption from the small intestine of indomethacin, bromthymol blue, sulphafurazole (sulfisoxazole), quinine, sulphanilic acid, phenol red (phenolsulfonphthalein), L-tryptophan and fluorescein isothiocyanate-dextrans. The effect of levamisole on the absorption from the small intestine of salicylic acid was marginally dose- and time-dependent, the maximal effect being observed after pretreatment with 2 mg of levamisole 1 day before the absorption studies. Sulphanilamide, similarly, was better sorbed from the small intestine and also from the stomach in the presence of levamisole. The intraperitoneal administration of levamisole may influence the absorption of some low molecular weight drugs from the gastrointestinal tract.

Animals↗

Renal handling of phenol red. II. The mechanism of substituted phenolsulphophthalein (PSP) dye transport in rabbit kidney tubules in vitro.

1. The uptake of various substituted phenolsulphophthalein dyes by cortical slices of rabbit kidney has been studied in detail in order to obtain more information on the secretory system for organic anions. 2. The rate of initial uptake of dyes and the accumulation after incubation for 2 hr under aerobic conditions increased in the order: phenol red (PR) greater than bromophenol blue (BPB) greater than bromocresol green (BCG) greater than bromothymol blue (BTB), while the reverse order of uptake was observed under anaerobic conditions. There was no difference between the uptake of BTB under aerobic and anaerobic conditions. 3. The accumulation of dyes under anaerobic conditions could be accounted for by binding to tissue constituents. In comparison with PR (Sheikh, 1972), the substituted dyes were found to interact extensively with the 700 G (cell membranes) and cytosol fractions of renal homogenates. 4. Low concentrations of the substituted dyes efficiently inhibited the accumulation of rho-aminohippurate (PAH). The concentration of dye resulting in 50% inhibition of PAH accumulation (KI) agreed well with concentrations estimated to sustain 50% of maximal dye transport (KM). On this basis the affinity of the dyes for the transport system increases in the order: PR less than BPB less than BCG less than BTB. 5. Probenecid, 2,4-dinitrophenol, PAH, octanoate and succinate affected to a smaller extent the uptake and binding of BPB and BCG by renal tissue than that previously shown for PR (Sheikh, 1972). No inhibitory effect of these substances on the accumulation of BTB by kidney tissue was observed. 6. The binding of PSP dyes by phospholipid vesicles (liposomes) and a representative binding protein, human serum albumin, exhibited close similarity to that of binding by renal tissue. Partition experiments involving octanol-water phases indicated that the hydrophobicity of the dyes increased in the order: PR less than BPB less than BCG less than BTB. 7. The results indicate that BTB, despite its inhibitory potency, is not transported by the organic anion system. BPB and BCG are transported to a lesser extent, and interact more strongly with the transport system than does PR. It is suggested that the substituted dyes by virtue of hydrophobic interaction with the transport system reduce the movement of the mobile part of the transport system.

Aerobiosis↗

New medium for improved recovery of coliform bacteria from drinking water.

A new membrane filter medium was developed for the improved recovery of injured coliforms from drinking water. The new medium, termed m-T7, consists of 5.0 g of Difco Proteose Peptone no. 3, 20 g of lactose, 3.0 g of yeast extract, 0.4 ml of Tergitol 7 (25% solution), 5.0 g of polyoxyethylene ether W-1, 0.1 g of bromthymol blue, 0.1 g of bromcresol purple, and 15 g of agar per liter of distilled water. Additional selectivity may be obtained by aseptically adding 0.1 microgram of penicillin G per ml to the medium after autoclaving. In laboratory studies, m-T7 agar recovered 86 to 99% more laboratory-injured coliforms than did m-Endo agar. m-T7 agar also recovered an average of 43% more verified coliforms from 67 surface and drinking water samples than did the standard m-Endo membrane filter technique. From drinking water, m-T7 agar recovered nearly three times more coliforms than did m-Endo agar. Less than 0.5% of the colonies on m-T7 agar gave false-negative reactions, whereas greater than 70% of the typical yellow colonies from m-T7 agar produced gas in lauryl tryptose broth. Most of the verified coliforms isolated on m-T7 agar belonged to one of the four common coliform genera: Escherichia, 17.6%; Klebsiella, 21.7%; Citrobacter, 17.3%; Enterobacter, 32.2%. The results demonstrate that m-T7 agar is superior to m-Endo agar, especially for the isolation of injured coliforms from drinking water.

Citrobacter↗

Selective medium for isolation of Xanthomonas maltophilia from soil and rhizosphere environments.

A selective medium (XMSM) was developed for isolation of Xanthomonas maltophilia from bulk soil and plant rhizosphere environments. The XMSM basal medium contained maltose, tryptone, bromthymol blue, and agar. Antibiotics added to select for X. maltophilia were cycloheximide, nystatin, cephalexin, bacitracin, penicillin G, novobiocin, neomycin sulfate, and tobramycin. A comparison was made between XMSM and 1/10-strength tryptic soy broth agar for recovery of X. maltophilia from sterile and nonsterile soil infested with known X. maltophilia isolates. A recovery rate of 97% or greater for XMSM was demonstrated. XMSM was used to isolate X. maltophilia from a variety of soil and rhizosphere environments.

Culture Media↗

Detection and enumeration of fecal indicator organisms in frozen sea foods. II. Enterococci.

Consistently high recoveries of enterococci as compared to the low numbers of coliforms obtained from the same samples of frozen sea foods are indirect evidence that enterococci are better indicators of contamination in such foods. The use of azide dextrose broth, modified by the incorporation of bromthymol blue, and of ethyl violet azide broth as presumptive and confirmation tests, respectively, were found to be highly specific for the detection and enumeration of enterococci in these samples. Tetrazolium agar medium, when used as a third step after the confirmation test, provides a reliable differentiation of Streplococcus faecalis types from other group D streptococci. A simple procedure is described for further identification of S. faecalis varieties and other enterococcal species. Incidence of biotypes within certain species is noted and relationships of these subgroups to the organisms described by other workers is discussed. The striking resistance of all group D streptococci to dihydrostreptomycin and polymyxin B seems to offer promise for evolving a new selective medium for these organisms.

Culture Media↗

Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.

Cryptococcus↗

Serotype B/C Cryptococcus neoformans isolated from patients in nonendemic areas.

Of 90 clinical isolates of Cryptococcus neoformans studied, 3 were determined to be serotype B/C. The patients from whom these B/C isolates were obtained were identified as never having lived in or visited the areas associated with B/C serotypes. This finding suggests a broader geographic distribution of this serotype group than previously believed. The glycine-cycloheximide-phenol red medium described by Salkin and Hurd (J. Clin. Microbiol. 15:169-171, 1982) was shown to be more accurate in differentiating A/D and B/D serotype pairs of C. neoformans than the creatinine-dextrose-bromthymol blue medium described by Kwon-Chung et al. (Int. J. Syst. Bacteriol. 28:616-620, 1978).

Cryptococcosis↗

Modified oxidation-fermentation medium for detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis.

A modified oxidation-fermentation medium was developed as a practical medium for highly sensitive and specific detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis. A total of 756 strains representing 17 Neisseria spp. and Branhamella catarrhalis were tested in this medium, in which the protein concentration was reduced relative to the carbohydrate concentration, phenol red was substituted for bromthymol blue at a low concentration, and the initial pH was adjusted to 7.2. Sugar utilization patterns were consistent with published results and with other cultural and biochemical characteristics for these species. The reactions obtained using this medium were qualitatively better and more reproducible than those obtained in cystine-Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) medium.

Acids↗

[Cryptococcus neoformans isolation from the soil in Panama].

Reports of Cryptococcus neoformans isolations from patients in Panama have appeared since 1978, but to our knowledge, thIs is the first report of isolation of this organism from soil samples in the Republic. Of 25 soil samples analyzed, two (25%) yielded the organism on birdseed agar medium. Canavanine-glycine-bromthymol blue medium served to identify the isolates as belonging to C. neorformans var neoformans, and not C. Neoformans var. gattii. Initially, isolates did not have capsules, but upon passage through newborn mice, wide capsules developed. This finding has important public health implications, since non-encapsulated environmental organisms, once in the host, can develop capsules, which significantly contribute to virulence.

Cryptococcus neoformans↗

[Legionella pneumophila in thermal pools of hot springs in the central Anatolian district].

Microbiological contamination of hot spring waters is a public health problem, and hot spring waters have been thought to be a potential source of Legionella infections. In this study, 209 water samples collected from 69 thermal pools of 36 hot springs in the Central Anatolia Region were examined for the presence of Legionella spp. between September and November 2001. The water samples were concentrated via filtration, and the filtrates were decontaminated by low-pH method. Then the samples were cultured on non-selective (Buffered Charcoal Yeast Extract Agar, BCYE) and selective (GVPC; BCYE supplemented with glycine, vancomycin, polymyxin B, cycloheximide, and MWY; BCYE supplemented with glycine, anisomycin, polymyxin B, vancomycin, bromthymol blue, bromcresole purple) media, and suspected colonies were confirmed by Legionella Latex Kit (Oxoid) and direct fluorescent antibody test. As a result, 24 out of 209 (11.5%) water samples were found positive for Legionella pneumophila, and a total 26 L. pneumophila strains were isolated from these 24 samples. Two of these isolates were found reactive with serogroup 1, 20 were reactive with serogroup 2-14 antisera in agglutination test, while 2 samples collected from the same thermal pool, have yielded 2 strains, of which one was serogroup 1 and the other was serogroup 2-14. L. pneumophila was detected in 22.2% of hot springs (8 of 36) and 14.5% of thermal pools (10 of 69). L. pneumophila concentrations which were detected in water samples ranged from 10 to 430 CFU/100 ml. This is the first data for the prevalence of Legionnaires' disease agent in hot spring spas in the Central Anatolia Region of Turkey. Colonization of bacterium in some spas indicates that the certain hot springs might be endemic focuses for Legionnaires' disease in our country.

Balneology↗

[Analysis of steroids. Part 45: Analytical investigation of pipecuronium bromide (Arduan)].

The following methods are described for the analytical investigation of pipecuronium bromide. 1. HPLC method. Of the several systems tried for the separation and quantification of impurities and degradation products the best results were obtained using silica as the stationary phase and 43:43:14 mixture of methanol, acetonitrile and concentrated aqueous ammonia containing 0.1 mole/l each of ammonium chloride and ammonium carbonate as the eluent. The validation of this method is presented. The above described aggressive eluent can be successfully replaced by an ion-pairing system using silica as the stationary phase and 96:4 mixture of acetonitrile and water containing 0.1 mole/l sodium perchlorate as the eluent. 2. Thin-layer chromatography. TLC systems are described for the separation and densitometric quantification of the impurities and degradation products of pipecuronium bromide. 3. Spectrophotometry. Two methods are described. The ester groups of the molecule can be determined by the iron(III)-hydroxamate method while for the ion-pair extraction of the quaternary ammonium steroid picric acid or bromthymol blue are used as the reagents. 4. Titrimetry. In addition to the titration with acetous perchloric acid for the assay of the bulk material a microtitration method is described for the determination of pipecuronium bromide in individual lyophylized ampoules (potentiometric titration with 0.1 M silver nitrate).

Androstane-3,17-diol↗

[Extraction-spectrophotometric determination of carbethopendecinium-bromide in drug preparations].

The paper elaborated the conditions for the determination of carbethopendecinium bromide with bromthymol blue and methyl orange (concentration of the drug and the colouring agent, pH of the aqueous phase, period of extraction with chloroform). Within a carbethopendecinium bromide concentration range of 1.10(-5) to 6.10(-5) mol/l, the linear dependence of absorbance of the ion-associate with the colouring agent on concentration was confirmed. At pH 3 and pH 10, the above-described procedure was used to determine the content of carbethopendecinium bromide in medicinal preparations (eye and nasal drops) using the calibration curve and comparison with one standard. The results were reproducible and the found deviations from the declared content ranged within a tolerance of +/- 10%.

Ophthalmic Solutions↗

Spectrophotometric determination of certain antidepressants in pharmaceutical preparations.

Simple and reproducible spectrophotometric methods have been developed for determination of sertraline, fluoxetine, and venlafaxine in pharmaceutical preparations. The methods are based on the reactions between the studied drug substances and ion-pair agents (bromothymol blue, bromocresol green, or bromophenol blue) to produce yellow-colored ion-pair complexes in acidic buffers. After extracting in chloroform, the ion-pair complexes are spectrophotometrically determined at the optimum wavelength. Optimizations of the reaction conditions were carried out. Beer's law was obeyed within the concentration range from 1 to 15 microg/mL. The molar absorptivity, Sandell sensitivity, and detection and quantification limits were also determined. The developed methods were applied successfully for the determination of these drugs in some available commercial preparations. The results were compared statistically with those obtained from reported high-performance liquid chromatography methods.

Antidepressive Agents↗

[The effect of the type of peptone for protein catabolism and realkalinization in Klebsielleae cultures].

According to the authors of the present paper, the formation of the MR+ and MR- groups is not explained by the variable degree of acidification, but by the various capacity of realkalinization of cultures. pH increases to values tending to 8 due to the formation of amines, ammonia and other basic products. They are the result of oxidative degradation processes, thus explaining the dependence on oxygen and the strictly aerobic character of the reaction. The acid-alkaline variation is influenced by the different content of peptones in monoaminodicarboacylic (meat-contraindicated) and diaminomenocarvoxylic (casein/soya bean-recommended) acids. The alkaline reversion may be determined in liquid media, having included ab initio either bromthymol blue, or bromcresol purple. The changes in pH may be read directly, colorimetrically, after an 18-20-hour incubation. By changing the interpretation (acid from glucose) and the indicator suggested by Clark and Lubs, the name "methyl red" becomes meaningless. At the same time, the introduction of other biochemical parameters (proteinic catabolism, realkalinization, dependence on oxygen) and the modification of the conditions of methods, justify the suggestion that the process of alkaline reversion, noted by the abbreviation "Reval", should be considered as a "per se" reaction and the laboratory determination should be patented as a routine test for the diagnosis of Enterobacteria.

Bacterial Proteins↗