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PubMed1995

Sensitivity of sandwich enzyme-linked immunosorbent assay for Cryptococcus neoformans polysaccharide antigen is dependent on the isotypes of the capture and detection antibodies.

Immunoglobulin M (IgM) and IgG1 monoclonal antibody isotype switch variants to Cryptococcus neoformans capsular polysaccharide were used to study the sensitivity of a double sandwich enzyme-linked immunosorbent assay (ELISA). The most sensitive ELISA configurations used IgG1 monoclonal antibody absorbed on polystyrene plates or IgM immobilized with goat antisera for antigen capture.

Animals↗
PubMed1995

Oxygen as limiting nutrient for growth of Cryptococcus neoformans.

In microbroth cultures with RPMI 1640 medium, the growth yield of seven Cryptococcus neoformans isolates was unaffected by augmentation of the normal (0.2%) glucose concentration in the medium to 2%, and the addition of other potential carbon, nitrogen, and vitamin sources to the medium also failed to produce large changes in growth yield. However, macrobroth cultures of C. neoformans in RPMI 1640 that were agitated by rotation in air gave turbidities 6 to 37 times greater than those in identical cultures incubated statically, and similar levels of increase were seen whether the medium contained 0.2 or 2% glucose. Incubation of microplates under an oxygen atmosphere or with agitation by rotation led to an increase of up to twofold in growth turbidity of the yeast. The maximum increase was achieved by incubation with rotation and was dependent on the brand of microplate used. The findings implicate oxygen as a growth-limiting nutrient for C. neoformans. Incubation of microbroth cultures under conditions that enhance oxygen availability for antifungal susceptibility testing purposes may increase the speed of such tests and enhance the determination of MIC endpoints.

Antifungal Agents↗
PubMed1995

Diversity of DNA fingerprints in Cryptococcus neoformans.

DNA fingerprint patterns of 156 Cryptococcus neoformans isolates (26 AIDS patients, 46 non-AIDS patients, and 40 environmental sources) from both varieties (126 C. neoformans var. neoformans and 30 C. neoformans var. gattii isolates) and from seven countries were analyzed by using the DNA probe UT-4p. Nine and twelve distinct DNA fingerprint patterns were observed for isolates of the C. neoformans var. neoformans and var. gattii, respectively. No pattern was unique to AIDS patients, non-AIDS patients, or the environment. Pattern II was observed more often in non-AIDS patients (8 of 23) than in AIDS patients (0 of 25). Pattern V was the most prevalent pattern (42 of 82) in clinical and environmental isolates. Isolates from three AIDS patients in Burundi and Zaire exhibited patterns identical to each other but different from those of isolates collected from their houses (i.e., dust of floors, walls, etc.) or a nearby pigeon coop. DNA fingerprint stability was determined for 53 isolates from nine non-AIDS patients at different time intervals during 5 to 128 weeks of antifungal therapy. For eight patients, the fingerprint pattern was stable while the ninth may have had a mixed infection. Pattern II was observed in 4 of 9 patients, which is similar to 4 of 14 in other non-AIDS patients as reported here. In spite of the extensive pattern heterogeneity among 15 C. neoformans var. gattii isolates in Australia, the patterns observed in seven California isolates were quite different from those in Australia. Among isolates of C. neoformans var. gattii, one fingerprint pattern (designated b) was observed in several countries of the Far East. The fingerprint patterns of two of three environmental isolates from Eucalyptus camaldulensis trees in Australia were identical to those of 2 of the 12 clinical isolates from the country.

AIDS-Related Opportunistic Infections↗
PubMed1995

Comparison of multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis for molecular subtyping of Cryptococcus neoformans. The Cryplococcal Disease Active Surveillance Group.

We evaluated multilocus enzyme electrophoresis (MEE) and random amplified polymorphic DNA (RAPD) for their usefulness in subtyping 344 Cryptococcus neoformans clinical isolates obtained from four U.S. metropolitan areas in 1992 to 1994. MEE and RAPD with five primers both discriminated between the two varieties of C. neofromans. MEE divided C. neoformans var. neoformans isolates into 15 enzyme electrophoretic subtypes (ETs) arranged in three complexes. The predominant ET 1 complex contained 10 ETs, with isolates from 70% of patients in 1 ET. RAPD with five primers further sorted this predominant ET into 19 subtypes, with 60% of isolates sorting into three RAPD types. The ET 8 MEE complex, containing three ETs, could not be divided further by RAPD. The ET 7 complex (two ETs) included isolates from all serotype AD patients. Although both MEE and RAPD identified isolates of C. neoformans var. gattii, neither distinguished between serotypes B and C. These results showed that the two C. neoformans varieties could be identified by MEE or RAPD profile as well as by biochemical methods. RAPD improved the discriminatory power of MEE for isolates within the ET 1 complex but with other ETs offered little additional sensitivity over MEE and was less sensitive than MEE with isolates of C. neoformans var. gattii. This information will be useful in identifying particular environmental sources of disease-causing exposures, in seeking clusters of cases, and in determining whether an infecting strain changes over time.

Cryptococcus neoformans↗
PubMed1996

Detection of Cryptococcus neoformans gene in patients with pulmonary cryptococcosis.

Pulmonary cryptococcosis was diagnosed by nested PCR. Extraction of DNA was performed by mechanical destruction of the capsules of Cryptococcus neoformans by the glass bead technique. Nested PCR was positive for 4 of 5 culture-positive specimens but negative for 1 culture-positive specimen, 10 culture-negative specimens, and 1 specimen with undetermined culture results.

Base Sequence↗
PubMed1996

Serotyping of Cryptococcus neoformans by dot enzyme assay.

A method is described for the serotyping of Cryptococcus neoformans based on direct analysis of culture supernatants for the major type-specific capsular antigen, glucuronoxylomannan. Factor sera prepared by absorption of polyclonal rabbit antisera (Iatron Laboratories, Inc., Tokyo, Japan) or selected anti-C. neoformans monoclonal antibodies were used in a dot enzyme assay to detect the presence of antigen.

Animals↗
PubMed1996

Molecular subtype distribution of Cryptococcus neoformans in four areas of the United States. Cryptococcal Disease Active Surveillance Group.

To improve understanding of the epidemiology of cryptococcal disease, we analyzed the multilocus genotype distribution of 358 Cryptococcus neoformans isolates obtained from 251 patients through active surveillance in four U.S. geographic areas from 1992 through 1994. Isolates of the predominant enzyme electrophoretic type (ET), ET-1, were recovered in significantly greater proportion from Atlanta, Ga., Houston, Tex., and all major metropolitan areas of Alabama than from San Francisco, Calif. ET-2 and ET-7 complex (serotype AD) isolates were recovered predominantly from San Francisco. ET-3 was recovered less frequently from San Francisco than from the three other locations. These findings may reflect geographic differences in exposure to environmental strains or the identification of previously unrecognized C. neoformans clusters. Analysis by random amplified polymorphic DNA-PCR subtyping further divided 67 ET-1 isolates into 19 additional subtypes, none of which could be associated with a particular geographic region. Multiple isolates from the same patient always revealed the same multilocus enzyme electrophoresis and random amplified polymorphic DNA subtypes. No differences in subtype distribution were found when isolates from AIDS patients were compared with those from persons without or with another underlying disease, although one C. noeformans var. gattii isolate was obtained from an AIDS patient. When body site distribution was analyzed, ET-4 was disproportionately recovered from skin or surface body sites. Evidence for linkage disequilibrium in this fungal population suggests that virulent C. neoformans possesses a clonal population structure. Continued application of molecular subtyping methods will be useful in tracking the source, transmission, and relative virulence of different C. neoformans strains.

AIDS-Related Opportunistic Infections↗
PubMed1996

Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR fingerprinting.

Sixty-one clinical and forty-nine environmental isolates of Cryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12- to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3'). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.

Australia↗
PubMed1996

Natural environmental sources of Cryptococcus neoformans var. gattii.

We sought evidence for new environmental sources of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA (RAPD) analysis of isolates from 29 animals with a restricted territorial range in five Australian states. Twenty-three of the 29 isolates and 45 of 45 eucalypt isolates tested previously exhibited one RAPD profile, VGI. RAPD profile VGII was identified in 6 of 17 isolates from domesticated species but in none of 12 native species. Four VGII isolates originated from an area of Western Australia with no natural stands of known eucalypt host, indicating the existence of at least one unrecognized natural source of C. neoformans var. gattii.

Animals↗
PubMed1996

Multicenter evaluation of broth microdilution method for susceptibility testing of Cryptococcus neoformans against fluconazole.

We have developed a microdilution method for measuring the susceptibility of Cryptococcus neoformans to fluconazole. The present study evaluated the interlaboratory agreement of the results for the microdilution method obtained at three different sites and compared this method with the National Committee for Clinical Laboratory Standards M27-P reference method. Excellent interlaboratory agreement among the results obtained at the three sites was achieved with this method (83 and 96% agreement within 1 and 2 log2 dilutions, respectively). An overall agreement of 90% between the microdilution method and the M27-P method was observed, demonstrating the comparability of the two methods. However, there are inherent problems with the M27-P method in relation to measuring C. neoformans susceptibility, including suboptimal growth of the organism in RPMI 1640, a longer incubation period, and a narrow range of MICs. On the basis of these data, the microdilution method tested in this study is recommended for inclusion in the National Committee for Laboratory Standards method for testing the antifungal susceptibility of C. neoformans.

AIDS-Related Opportunistic Infections↗
PubMed1996

Karyotype instability in Cryptococcus neoformans infection.

The electrophoretic karyotypes of 32 clinical and 3 environmental Cryptococcus neoformans isolates from New York City were studied by contour clamped homogeneous electrophoresis. There was extensive variation among the electrophoretic karyotypes of isolates from different patients. Sequential C. neoformans isolates from patients with chronic or relapsing infection had very similar karyotypes. However, minor differences in electrophoretic karyotypes were detected among sequential isolates from 50% of the patients studied, suggesting the occurrence of chromosomal rearrangements or deletions in vivo. This hypothesis was tested by infecting mice, recovering isolates from mouse organs, and comparing the electrophoretic karyotypes before and after passage. Three clinical and three environmental strains were studied before and after passage in mice. Karyotype differences were detected after mouse passage for one clinical and two environmental strains. Our results indicate (i) extensive karyotype variation among isolates from a small geographic regions, (ii) a high frequency of electrophoretic karyotype differences among sequential isolates from individual patients, and (iii) the occurrence of electrophoretic karyotype changes during experimental infection of mice. The implications of these observations are discussed.

AIDS-Related Opportunistic Infections↗
PubMed1996

Persistence, replacement, and microevolution of Cryptococcus neoformans strains in recurrent meningitis in AIDS patients.

Six separate human immunodeficiency virus-positive patients with cryptococcal meningitis were each found to have been infected with a unique strain of Cryptococcus neoformans on the basis of genomic DNA finger-printing analysis with the microsatellite sequence-containing oligonucleotide probe (GGAT)4 and by random amplification of polymorphic DNA. Two patients (A and B) experienced a recurrent episode of infection. Between 12 and 16 single-colony isolates recovered from primary isolation media (> 50% of C. neoformans colonies recovered) from cerebrospinal fluid specimens were fingerprinted from both patients during each episode. The fingerprints of both isolate collections from patient B were very similar, although minor polymorphisms were evident in both sets of profiles. The fingerprints of the isolate collection from the initial episode of infection in patient A were also identical to each other, apart from minor polymorphisms, but they were clearly different from the corresponding profiles of the isolate collection from the recurrent episode, the latter of which were completely identical, apart from minor polymorphisms in a single isolate. Furthermore, prolonged storage and in vitro subculture of the isolates did not alter the fingerprint profiles. These results provided convincing evidence that patients A and B were each infected with a single C. neoformans strain during each episode of infection and that in patient B, the same strain persisted and caused both episodes, while in patient A, a different strain was responsible for each episode. The prevalence of polymorphisms in multiple single-colony isolates from both patients also suggested that C. neoformans populations may undergo microevolution.

AIDS-Related Opportunistic Infections↗
PubMed1996

Modified India ink preparation for Cryptococcus neoformans in cerebrospinal fluid specimens.

A novel modified India ink technique for the diagnosis of Cryptococcus neoformans in cerebrospinal fluid specimens is described. It employs 2% chromium mercury and India ink. This technique allows a clear identification of some external and internal structures of the organism. Three layers from the outer capsule that have previously been discerned only by electron microscopy are distinguished.

Carbon↗
PubMed1997

DNA typing suggests pigeon droppings as a source of pathogenic Cryptococcus neoformans serotype D.

The unusual proportion of serotype D strains of Cryptococcus neoformans infecting patients diagnosed with cryptococcosis in some parts of France prompted the analysis of DNA fingerprints obtained with 26 clinical and 29 environmental isolates from the same area. Our results suggest that pigeon droppings are a potential source of pathogenic strains of C. neoformans serotype D, as previously demonstrated for serotype A.

Adult↗
PubMed1997

Isolation of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in India.

Cryptococcus neoformans var. gattii has an ecological association with five Eucalyptus species: E. blakelyi, E. camaldulensis, E. gomphocephala, E. rudis, and E. tereticornis. After human infections due to C. neoformans var. gattii were diagnosed in the states of Punjab, Himachal Pradesh, and Karnataka, India, a study was undertaken to investigate the association of C. neoformans var. gattii with Indian eucalypts, especially in the state of Punjab. A total of 696 specimens collected from E. camaldulensis, E. citriodora and E. tereticornis (hybrid) trees were examined for the presence of C. neoformans var. gattii. Flowers from two trees of E. camaldulensis in the Chak Sarkar forest and one from the village of Periana near the Ferozepur area yielded five isolates of C. neoformans var. gattii. The origin of the trees could be traced to Australia, thus providing evidence that the distribution of E. camaldulensis correlated with the distribution of human cryptococcosis cases caused by C. neoformans var. gattii in northern India.

Animals↗
PubMed1997

Genetic relatedness and diversity of Cryptococcus neoformans strains in the Maltese Islands.

The genetic relatedness of clinical and environmental Cryptococcus neoformans strains in the Maltese Islands was investigated by randomly amplified polymorphic DNA fingerprinting with four primers. The clinical strains isolate over the course of 1 year from AIDS patients showed identical fingerprints. The electrophoretic patterns of the two clinical strains were also the most common patterns among the environmental strains, but the patterns among the environmental strains showed a wide variability and no correlation with the site of isolation.

AIDS-Related Opportunistic Infections↗
PubMed1997

Molecular epidemiology of Cryptococcus neoformans in Brazil and the United States: evidence for both local genetic differences and a global clonal population structure.

Little is known about the global molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans. We studied 51 clinical and environmental (pigeon excreta) isolates from two cities in Brazil (Belo Horizonte and Rio de Janeiro) by analyzing their carbon assimilation patterns, electrophoretic karyotypes, restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element-1 (CNRE-1), and URA5 sequences. Results were compared to those previously obtained for isolates from New York City by the same DNA typing methods. Computer-assisted analysis of RFLPs and contour-clamped homogeneous electrophoresis (CHEF) patterns and URA5 sequences was performed to generate dendrograms. Some environmental and clinical isolates were found to be indistinguishable by CHEF, CNRE-1 RFLP, and URA5 sequence analyses. Similarly, some isolates from Rio de Janeiro and Belo Horizonte were indistinguishable by the three DNA typing techniques. Overall, Brazilian isolates appeared to be less heterogeneous by DNA analysis than isolates from other regions. Several Brazilian isolates were highly related to New York City isolates. Phylogenetic analysis of the sequences obtained for the Brazilian isolates and those obtained for New York City isolates was congruent with the dendrogram generated from the CNRE-1 RFLP data. In summary our results indicate (i) that the discriminatory power of the DNA typing method differs for Brazilian and New York City strains, with the order being CNRE-1 RFLP analysis > URA5 sequence analysis > CHEF analysis and CHEF analysis > URA5 sequence analysis > CNRE-1 RFLP analysis, respectively; (ii) that there are differences in local genetic diversity for Brazilian and New York City isolates; (iii) that there is additional evidence linking clinical isolates to those in pigeon excreta; and (iv) that some isolates from Brazil and New York City are closely related, consistent with the global dispersal of certain pathogenic strains.

Animals↗
PubMed1998

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.

Amphotericin B↗