Cryptococcal capsular polysaccharide clearance in nonimmune mice.

Clearance of cryptococcal polysaccharide (CP) from tissues and body fluids of nonimmune mice was studied. Mice were injected intravenously only with one mg of purified CP, and serum, urine and tissues were obtained from each animal at various intervals for a period of 84 days. Tissue extracts, serum and urine were tested for CP content by enzyme-linked immunosorbent assay (ELISA) and latex agglutination. High concentrations of CP were detected by both assays one-half hour after injection in blood (serum), liver, spleen, kidney and lung (extracts). The duration of ELISA detectable CP was longest (70 days) in liver and spleen and shortest (14 days) in lung extract. By 14 days after injection, concentration of CP in the blood fell below that found in the liver and spleen. CP remained detectable (titers 32-64) after all other extracts became negative. These results indicate that CP is stored in tissues (binding mechanism and site unknown), and that the liver and spleen possess greater storage capacity than other tissues. Antibody (IgM) to CP appeared in low titer on the 14th day and thereafter.

Enzyme-linked immunosorbent assays in murine cryptococcosis.

In this investigation, enzyme-linked immunosorbent assay (ElISA) procedures were used to study the time of appearance and the duration of demonstrable antigen and antibody in body fluids of mice with disseminated cryptococcosis. The ELISA antigen procedure detected cryptococcal capsular polysaccharide (CCP) in the serum and urine of infected mice 3 days after infection--4 days before it could be demonstrated by the latex agglutination procedure. ELISA-reactive antibody was present throughout the course of infection (mean death time, 32 days), whereas antibody was not detected by whole cell agglutination after day 20. High serum concentrations of CCP (titers to 64,000) persisted throughout the course of infection, while antibody declined to low levels with progression of disease. ELISA provides a sensitive system for quantitation and monitoring of antigen (CCP) processing and clearance (or storage), and for cryptococcal antibody formation in progressive cryptococcosis.

Cryptococcus neoformans serotype groups encountered in Oklahoma.

Eighty clinical and 28 soil isolates of C. neoformans obtained in Oklahoma were separated into A-D and B-C serotype groups utilizing creatinine-dextrose agar with bromthymol blue. Previously, serotype B-C clinical isolates have been frequent only in patients from Southern California where as many as 50% of the isolates are of this type. In contrast, in patients from the rest of the United States the B-C frequency has been only 6%. Of the 80 C. neoformans isolates from Oklahoma patients, 12 (15%) were serotype B-C. One-half of these 12 Oklahoma patients with serotype B-C isolates had no history of any travel to California, and were long-time residents of Oklahoma. All 28 soil isolates of C. neformans from Oklahoma in this study were serotype A-D. Since serotype B-C recovery from a soil sample has never been reported, attempts are in progress to isolate serotype B-C from the environments of these patients from Oklahoma.

Comparison of enzyme immunoassay and latex agglutination methods for detection of Cryptococcus neoformans antigen.

An enzyme immunoassay for the measurement of cryptococcal capsular polysaccharide in human body fluids is described. The enzyme immunoassay detects cryptococcal capsular polysaccharide at a concentration of 6 ng/ml, compared with 35 ng/ml detectable by the latex agglutination test. The enzyme immunoassay detects cryptococcal capsular polysaccharide in body-fluid specimens that are negative by the latex agglutination test. Titers by enzyme immunoassay are generally higher and persist longer into the treatment period than those determined by latex agglutination. No cryptococcal capsular polysaccharide is detected by the enzyme immunoassay procedure in fluids from subjects not known to have cryptococcosis. The enzyme immunoassay procedure presented here provides earlier detection of cryptococcal material in body fluids, and thereby diagnosis and treatment of cryptococcosis can be made earlier in the course of disease.

Development of an enzyme immunoassay for cryptococcal antibody.

An enzyme immunoassay (ELISA) for measurement of cryptococcal IgG antibody in human serum is described. Clinical studies indicate that the assay is a useful addition to the currently available techniques for measuring antibodies in cryptococcosis. IgG-specific antibody (titers 4 to 1,024) was detected in the serum of 78% of the cryptococcosis patients tested and in 61% of the serum from healthy individuals with positive delayed skin hypersensitivity to cryptococcin. The micro-ELISA for cryptococcal antibody is of potential value in patient management, and in epidemiological studies.

Rapid presumptive identification of Cryptococcus neoformans.

Carbohydrate-containing extracts were prepared from mature yeast colonies grown on Sabouraud dextrose agar by mixing a 0.001-ml loopful of yeast cells for 30 s in phenolized saline and removing the cells by centrifugation. Extracts were prepared from 54 Cryptococcus neoformans isolates, 29 isolates of other Cryptococcus species, 16 isolates of Candida species, 2 Rhodotorula, 2 Torulopsis, and 1 Saccharomyces species. Initially the carbohydrate content of each extract was estimated (Molisch method) and adjusted to 1, 5, and 10 microgram/ml. Twofold dilutions of each extract were tested for reactivity with the cryptococcal latex agglutination reagent of Bloomfield et al. (N. Bloomfield, M.A. Gordon, and D.F. Elmendorf, Jr., Proc. Soc. Exp. Biol. Med. 114:64-67, 1963). All 54 C. neoformans extracts gave strong agglutinations (3+ to 4+) in dilutions of 1:4 or greater. None of the other yeasts produced any agglutination, except for 1 of 15 C. laurentii isolates, which showed a 1+ reaction that disappeared at a dilution of 1:4 and above. Subsequent testing established that a single extract made from 0.001 ml of yeast cells in 6 ml of phenolized saline contained less than 5 microgram of carbohydrate per ml, was suitable for a single rapid screening dilution, and eliminated any cross-reaction from the C. laurentii isolates. In our hands this method has provided a reliable differentiation of C. neoformans from other unknown yeast colonies in less than 20 min exclusive of a Molisch determination.

Quantitation of microorganisms in sputum.

A method of quantitating microbial cultures of homogenized sputum has been devised. Possible application of this method to the problem of determining the etiologic agent of lower-respiratory-tract infections has been studied to determine its usefulness as a guide in the management of these infections. Specimens were liquefied by using an equal volume of 2% N-acetyl-L-cysteine. The liquefied sputum suspension was serially diluted to 10(-1), 10(-3), 10(-5), and 10(-7). These dilutions were plated on appropriate media by using an 0.01-ml calibrated loop; they were incubated and examined by standard diagnostic methods. Quantitation of fresh sputum from patients with pneumonia prior to antimicrobial therapy revealed that probable pathogens were present in populations of 10(7) organisms/ml or greater. Normal oropharyngeal flora did not occur in these numbers before therapy. Comparison of microbial counts on fresh and aged sputum showed that it is necessary to use only fresh specimens, since multiplication or death alters both quantitative and qualitative findings. Proper collection and quantitative culturing of homogenized sputum provided information more directly applicable to patient management than did qualitative routine methods. Not only was the recognition of the probable pathogenic organism in pneumonia patients improved, but serial quantitative cultures were particularly useful in recognizing the emergence of superinfections and in evaluating the efficacy of antimicrobial therapy.