Isolation of specific DNA probes from Cryptococcus neoformans

OBJECTIVE: To construct plasmid library and screen specific DNA probes for Cryptococcus neoformans. METHODS: Serotype A Cryptococcus neoformans was used as the study strain, plasmid pUC18 as vector, and Escherichia coli JM103 as host cell. The plasmid library of cryptococcus neoformans was constructed (pCN). Other pathogenes causing affection diseases which should be distinguished from cryptococcusis clinically, and other fungi similar to Cryptococcus neoformans with physiological and biochemical characteristics were used as a distinguishing system, specific colonies were screened by hibridization in double steps. RESULTS: The inserts of the library were 280 to 1800 base pairs and 580 base pairs in average length. Repeated sequence was 32.43% and single copy sequence was 67.57% in genome of cryptococcus neoformans respectively. Three specific colonies were isolated from the library. Colony pCNII A6 was serotype A specific, pCNII B5 species specific and pCNIII G1-specific for var. neoformans. CONCLUSION: A rapid diagnosis of Cryptococcus neoformans infection at early stage can be made by using species-specific probe, and serotype and variaty of neoformans and gattii be distinguished in epidemic study.     

Serial isolates of Cryptococcus neoformans from patients with AIDS differ in virulence for mice

Serial isolates of Cryptococcus neoformans from patients with chronic infection can exhibit minor karyotype changes as a result of chromosome length polymorphism (CLP). This study investigated whether serial C. neoformans isolates with CLP from 4 patients with AIDS exhibited biologic and phenotypic differences. CLP permits the identification of serial isolates in murine mixed infection. The parameters studied were virulence in mice, capsule size, colony morphology, melanization, protease production, MICs of antifungal drugs, and growth rates in vitro. Two parameters of virulence in mice were studied: persistence in tissue and survival time after lethal infection. Serial C. neoformans isolates were shown to differ in ability to persist in vivo, virulence in a murine infection model, in vitro growth rates at 37 degreesC, and capsule size. Melanin and protease production and MICs of antifungal drugs were comparable for serial isolates. These observations suggest microevolution of C. neoformans during human infection. This process may allow the fungal population to change, escape eradication by the immune system, and thus cause chronic infections.     

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.     

Dominant selection system for use in Cryptococcus neoformans

BACKGROUND: Approximately one-fourth of patients who could benefit from bone marrow transplantation (BMT) are served by a genotypically identical sibling donor. When patients do not have an HLA-matched donor, alternative marrow sources should be explored. The way to allow survival in some patients is to perform two- or three loci HLA mismatched BMT. Preliminary results with BMT in partially-matched, related donors performed at Veterans General Hospital-Taipei are reported. METHODS: Between 1985 and 1994, a total of 121 patients were enrolled in this study. Ten patients received BMT with HLA partially-matched, related donor, including 2 acute nonlymphocytic leukemia (ANLL), 5 chronic myelocytic leukemia (CML) and 3 severe aplastic anemia (SAA). Three and four hematologically malignant patients received cyclophosphamide (Cy) + radiotherapy (RT) and Cy + Busulfan(Bu) preconditioning regimens, respectively; three SAA patients received standard Cy + RT regimen. Additional prophylaxis against graft-versus-host disease (GVHD) consisted of methotrexate (MTX) and cyclosporin-A (CSA). The median follow up was 36 months. Seven were 2-loci disparate and 3 were 3-loci. RESULTS: Engraftment developed with a mean of 20.9 days after transplant. Nonengraftment rate was 1/10 (10%), delayed graft failure 2/10(20%) and venoocculsive disease (VOD) 1/10(10%). The percentage of patients who developed grade II to IV acute GVHD was low (13.6% of those mismatched at 0 locus, 31.6% mismatched at 1 loci and 14.3% at > or = 2 loci. p = 0.181). Extensive chronic GVHD occurred in 16.7% (34.1% of those mismatched at 0 locus, 41.2% mismatched at 1 loci and 16.7% at 2 loci. p = 0.492). There were five deaths. The other 5 still survived at 36 months of follow-up. Log-rank analysis revealed no statistical significance between those mismatched at > or = 2 vs at 1 (p = 0.146) but the difference between those mismatched at > or = 2 and at 0 (p = 0.0359) was statistically significant. CONCLUSIONS: When patients requiring BMT without an HLA identical sibling donor, an alternative transplant from haploidentical family members remains a viable option, especially when a patient has CML, SAA or other refractory hematologic malignancies.     

Pathogenesis of pulmonary Cryptococcus neoformans infection in the rat

The pathogenesis of Cryptococcus neoformans pulmonary infection in the rat was studied after intratracheal inoculation. Lungs were examined at various times following infection for histopathology in conjunction with macrophage markers, proliferating cell nuclear antigen (PCNA), and capsular glucuronoxylomannan (GXM) antigen. Serum GXM, immunoglobulin M (IgM) and IgG titers and organ fungal burden were compared with pathological findings. C. neoformans organisms were in the lung parenchyma 2 h postinoculation, and GXM antigen was present in surrounding tissues shortly thereafter. Extrapulmonary dissemination occurred early in infection. Two phases of host cellular inflammatory response were discernible: early local macrophage recruitment at 2 to 4 days followed by granulomatous inflammation, which reached maximum intensity 14 days after infection. The granulomatous phase was preceded by lymphocyte influx with macrophage proliferation and maturation into epithelioid histiocytes; this was paralleled by a shift of yeasts from extracellular to intracellular spaces. Tissue IgG deposits, serum IgG to GXM, and localization of tissue GXM immunoreactivity to epithelioid cells were noted at 2 to 4 weeks. A 10-fold decrease in lung fungal burden occurred 25 days postinfection and was associated with resolving granulomas, fewer proliferating cells, and decreased tissue GXM. The present study demonstrates that (i) C. neoformans penetrates the lung parenchyma shortly after infection; (ii) immunocompetent rats control pulmonary cryptococcosis efficiently, with minimal extrapulmonary dissemination and low levels of serum GXM; and (iii) macrophage activation is likely to play a crucial role in limiting C. neoformans infection in the rat lung.     

Bivalency is required for anticapsular monoclonal antibodies to optimally suppress activation of the alternative complement pathway by the Cryptococcus neoformans capsule

Encapsulated cells of Cryptococcus neoformans are potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)2 and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)2 fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.     

A rapid urease test for presumptive identification of Cryptococcus neoformans

Extra-contractual referrals (ECRs) can be a cause of considerable anxiety to purchasing authorities, mainly because of their potential to generate unexpected expenditure. But ECRs can also be used as a tool for monitoring the demand for, and quality of, clinical services. ECRs were studied in the Darlington Health Authority district using a variety of methods including inter-disciplinary meetings, a series of interviews with local GPs, and a questionnaire to general practitioners on 230 consecutive ECRs. The methods and results of the questionnaire study are presented. The commonest reasons for making ECRs included the mistaken belief that a contract existed with the ECR provider, patient dissatisfaction with the local provider, and referral to benefit from shorter waiting lists. ECRs for bone-mass densitometry, orthopaedics, and ear nose and throat services were over-represented. Questionnaire results were validated by comparison with an interview study of all GPs in the district. We conclude that trends in ECRs can be monitored as a convenient "early warning system' to alert purchasing authorities to changes in demand or perceived problems with local provider units. ECR data must be interpreted in the context of further local background information from sources such as GPs and public health physicians. In the case of Darlington, scrutiny of ECRs has led to changes in services and contracts.     

Phenotypic switching in the human pathogenic fungus Cryptococcus neoformans is associated with changes in virulence and pulmonary inflammatory response in rodents

High-frequency reversible changes in colony morphology were observed in three strains of Cryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as 10(-3). Mice infected with colony type W died faster than those infected with other colony types. The rat inflammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inflammation with caseous necrosis for infection with type C to minimal inflammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains of C. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate that C. neoformans undergoes phenotypic switching and that this process can affect virulence and host inflammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.     

Structural characterization of the galactoxylomannan of Cryptococcus neoformans Cap67

The galactoxylomannan (GalXM) obtained from the culture supernatant of an acapsular mutant of Cryptococcus neoformans Cap67 was purified by Concanavalin A affinity, ion-exchange, and gel-filtration chromatographies. The structure of GalXM was determined by methylation analysis and by 1D and 2D NMR spectroscopic studies of the intact polysaccharide and of the oligosaccharide fragments generated by Smith degradation and by acetolysis. GalXM is a complex polysaccharide with an alpha-(1-->6) -galactan backbone. The polysaccharide is branched at c-3 of alternate Gal units of the backbone. C-3 is the point of attachment of the oligosaccharide side chains comprised of alpha-D-Man- (1-->3)-alpha-D-Man-(1-->4)- beta-D-Gal-substituted with zero to three terminal beta-Xyl residues as shown in the following structure: [formula: see text].     

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 microM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.     

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

PURPOSE: Pilocarpine, a salivary stimulant, has been shown to protect salivary glands from gamma-radiation-induced damage during the radiotherapy of head and neck tumors. This study was performed to determine whether pilocarpine affects the survival of squamous carcinoma cells, line SCC-25, following gamma-radiation treatment. METHODS AND MATERIALS: The survival of squamous carcinoma tumor cells, line SCC-25, following the exposure of cells to pilocarpine at concentration of 0-100 ng/ml given for 0-1 h prior to radiation at dose of 0-20 Gy was determined by an in vitro colony-formation assay. RESULTS: The survival fractions of SCC-25 cells were identical for the control and pilocarpine-treated samples at all tested conditions. Calculated Do and Dq values did not depend on the presence of pilocarpine and were not affected by the time of incubation prior to irradiation. CONCLUSION: Pilocarpine, at clinically relevant concentrations, given to the SCC-25 cells 1 h prior to or at the time of irradiation did not affect survival of SCC-25 cells in vitro. Pilocarpine does not sensitize or protect these tumor cells from the effects of y-radiation, suggesting that this agent should not compromise the tumoricidal effects of radiotherapy.     

Susceptibility testing of Cryptococcus neoformans using the urea broth microdilution method

An urea broth microdilution method to assay the susceptibility of Cryptococcus neoformans to antifungal drugs was newly developed. Using this method, urease activity of the fungus was measured instead of the viability by checking colony development. The urease activities were indicated by colour changes in optical density at 545 nm. The end point in this assay was considered as 99% inhibitory concentration. When we measured antifungal activities of the three drugs against 16 isolates of Cr. neoformans using this assay method, mean minimum-inhibitory concentrations (MICs) of fluconazole, itraconazole and terbinafine were 2.0 micrograms ml-1, 0.008 microgram ml-1 and 0.25 microgram ml-1 respectively. This assay method resulted in higher sensitivity in MICs of the three antifungal drugs than the broth microdilution method recommended by the Committee for Laboratory Standards of the Japanese Society for Medical Mycology. The results obtained using this assay method support the more effective evaluation of antifungal substances in susceptibility testing of Cr. neoformans.     

Comparison of broth macrodilution, broth microdilution and E-test susceptibility tests of Cryptococcus neoformans for fluconazole

Forty Cryptococcus neoformans strains isolated from cerebral spinal fluid specimens collected from 39 patients were included in the study. The MICs for fluconacole were determined by YNB macrodilution test, microdilution tests using both RPMI1640 and YNB medium and E-tests on solidified RPMI1640 medium, Casitone and YNB agar. In comparison with the reference macrodilution method NCCLS M27-P both the microdilution as well as the E-test techniques can be used for fluconacole susceptibility testing of Cr. neoformans.     

The vacB gene required for virulence in Shigella flexneri and Escherichia coli encodes the exoribonuclease RNase R

vacB, a gene previously shown to be required for expression of virulence in Shigella and enteroinvasive Escherichia coli, has been found to encode the 3'-5' exoribonuclease, RNase R. Thus, cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy of vacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacB gene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacB be renamed rnr.     

Detection rate of Cryptococcus neoformans in cerebrospinal fluid specimens at Kenyatta National Hospital, Nairobi

A ten-year retrospective review of laboratory detection of Cryptococcus neoformans in cerebrospinal fluid was undertaken using past laboratory and clinical records at Kenyatta National Hospital. A total of 1462 India-ink tests were carried out, 76 (5.2%) of these tested positive for C. neoformans. An increasing number of clinical requests for India-ink test mirrored by increasing number of patients with immunological disorders were noted over the study period although no obvious trend emerged in the detection pattern of C. neoformans. The use of a more sensitive test such as the latex agglutination technique is suggested.     

Encapsulation of Cryptococcus neoformans with glucuronoxylomannan inhibits the antigen-presenting capacity of monocytes

PURPOSE: The combination of carmustine (BCNU), dacarbazine (DTIC), cisplatin (DDP), and tamoxifen (Tam) has been reported in small series to provide a response rate of 50%, but with significant myelosuppression and risk of thromboembolic complications. We performed this phase II study to assess the antitumor activity and important toxicities of this combination in the cooperative group setting. PATIENTS AND METHODS: Seventy-nine eligible patients were treated with BCNU 150 mg/m2/d, every 6 weeks, DTIC 220 mg/m2/d on days 1 to 3 every 3 weeks, DDP 25 mg/m2/d on days 1 to 3 every 3 weeks, and Tam 20 mg orally daily throughout treatment. Treatment cycles were repeated every 6 weeks in responding or stable patients for a maximum duration of 1 year. RESULTS: Twelve objective responses were achieved (response rate 15%, 95% confidence interval 8%-25%). Five responses were complete (CR) and seven were partial (PR). The median response duration was 8+ (range, 4-19+) months, (16+ [4-19+] for CR and 8+ [4-11] for PR), and the median survival of the entire group was 9 months. The toxicities were predominantly neutropenia and thrombocytopenia. Four patients developed thromboembolic events. Two patients died while on protocol therapy, one with complications of neutropenia, and the other with disease progression. CONCLUSION: The activity of this regimen is in the range reported for single agents or DTIC plus DDP, and the addition of BCNU and Tam appears to increase toxicity. We do not recommend this combination for routine treatment of advanced melanoma or as the control arm in randomized studies of combination therapy.     

An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum

Ralstonia solanacearum, which causes bacterial wilt disease of many plant species, produces several extracellular plant cell wall-degrading enzymes that are suspected virulence factors. These include a previously described endopolygalacturonase (PG), PehA, and two exo-PGs. A gene encoding one of the exo-PGs, pehB, was cloned from R. solanacearum K60. The DNA fragment specifying PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa with a typical N-terminal signal sequence. The cloned pehB gene product cleaves polygalacturonic acid into digalacturonic acid units. The amino acid sequence of pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2% identity at the amino acid level. PehB also has limited similarity to plant exo-PGs from Zea mays and Arabidopsis thaliana. The chromosomal pehB genes in R. solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced with an insertionally inactivated copy of pehB. The resulting mutants were deficient in the production of PehB and of both PehA and PehB, respectively. The pehB mutant was significantly less virulent than the wild-type strain in eggplant virulence assays using a soil inoculation method. However, the pehA mutant was even less virulent, and the pehA pehB double mutant was the least virulent of all. These results suggest that PehB is required for a wild-type level of virulence in R. solanacearum although its individual role in wilt disease development may be minor. Together with endo-PG PehA, however, PehB contributes substantially to the virulence of R. solanacearum.     

Efficacy of NS-718, a novel lipid nanosphere-encapsulated amphotericin B, against Cryptococcus neoformans

In vitro and in vivo efficacies of NS-718, a lipid nanosphere-encapsulated amphotericin B (AMPH-B), have been studied. Of the tested AMPH-B formulations, NS-718 had the lowest MIC for Cryptococcus neoformans. In a murine model, low-dose therapy (0.8 mg/kg of body weight) with NS-718 showed higher efficacy than that with AmBisome. High-dose therapy (2.0 mg/kg) with NS-718 was much more effective than those with Fungizone and AmBisome. In mice treated with a high dose of NS-718, only a few yeast cells had grown in lung by 7 days after inoculation. A pharmacokinetic study showed higher concentrations of AMPH-B in lung following administration of NS-718 than after administration of AmBisome. Our results indicated that NS-718, a new AMPH-B formulation, is a promising antifungal agent for treatment of pulmonary cryptococcosis and could be the most effective antifungal agent against C. neoformans infections.     

Isolation and characterization of a novel connexin gene, Cx-60, in porcine ovarian follicles

Sequences present at the genomic termini of herpesviruses become linked during lytic-phase replication and provide the substrate for cleavage and packaging of unit length viral genomes. We have previously shown that homologs of the consensus herpesvirus cleavage-packaging signals, pac1 and pac2, are located at the left and right genomic termini of human herpesvirus 6 (HHV-6), respectively. Immediately adjacent to these elements are two distinct arrays of human telomeric repeat sequences (TRS). We now show that the unique sequence element formed at the junction of HHV-6B genome concatemers (pac2-pac1) is necessary and sufficient for virally mediated cleavage of plasmid DNAs containing the HHV-6B lytic-phase origin of DNA replication (oriLyt). The concatemeric junction sequence also allowed for the packaging of these plasmid molecules into intracellular nucleocapsids as well as mature, infectious viral particles. In addition, this element significantly enhanced the replication efficiency of oriLyt-containing plasmids in virally infected cells. Experiments revealed that the concatemeric junction sequence possesses an unusual, S1 nuclease-sensitive conformation (anisomorphic DNA), which might play a role in this apparent enhancement of DNA replication--although additional studies will be required to test this hypothesis. Finally, we also analyzed whether the presence of flanking viral TRS had any effect on the functional activity of the minimal concatemeric junction (pac2-pac1). These experiments revealed that the TRS motifs, either alone or in combination, had no effect on the efficiency of virally mediated DNA replication or DNA cleavage. Taken together, these data show that the cleavage and packaging of HHV-6 DNA are mediated by cis-acting consensus sequences similar to those found in other herpesviruses, and that these sequences also influence the efficiency of HHV-6 DNA replication. Since the adjacent TRS do not influence either viral cleavage and packaging or viral DNA replication, their function remains uncertain.     

In vitro and in vivo efficacy of the triazole TAK-187 against Cryptococcus neoformans

Multiple isolates of Cryptococcus neoformans, including those with fluconazole resistance, were tested to assess the in vitro activity of the new triazole TAK-187. MICs of TAK-187 were at least eightfold lower than those of fluconazole, and fungicidal concentrations for most isolates were 4 microg/ml or less. TAK-187 also was evaluated as intermittent therapy using two dosages in a rabbit model of experimental cryptococcal meningitis. Compared to daily treatment with fluconazole, as little as two doses of TAK-187 given 7 days apart were found to be effective. Plasma and cerebrospinal fluid TAK-187 concentrations were many times higher than MICs and fungicidal concentrations. Based upon its therapeutic efficacy and long half-life in the rabbit model, TAK-187 should be investigated for intermittent dosing in treatment or suppression of cryptococcal infections in humans.