Diagnosis and inflammatory response of patients with candiduria.

Candiduria is common in hospitalised patients, but the clinical relevance is still unclear. This study was done to further our knowledge on detection of and host responses to candiduria. Urines and clinical data from 136 patients in whom presence of yeast was diagnosed by microscopic urinalysis were collected. Diagnosis by standard urine culture methods on blood and MacConkey agar as well as on fungal culture medium (Sabouraud dextrose agar) was compared. Inflammatory parameters (IL-6 and IL-17, Ig) were quantified in the urine and compared with levels in control patients without candiduria. Standard urine culture methods detected only 37% of Candida spp. in urine. Sensitivity was especially low (23%) for C. glabrata and was independent of fungal burden. Candida specific IgG but not IgA was significantly elevated when compared with control patients (P < 0.0001 and 0.07 respectively). In addition, urine levels of IL-6 and IL-17 were significantly higher in candiduric patients when compared with control patients (P < 0.001). Multivariate analysis documented an independent association between an increased IgG (odds ratio (OR) 136.0, 95% confidence interval (CI) 25.7-719.2; P < 0.0001), an increased IL-17 (OR 17.4, 95% CI 5.3-57.0; P < 0.0001) and an increased IL-6 level (OR 4.9, 95% CI 1.9-12.4; P = 0.001) and candiduria. In summary, our data indicate that clinical studies on candiduria should include fungal urine culture and that inflammatory parameters may be helpful to identify patients with clinically relevant candiduria.

Cryptococcus neoformans variants generated by phenotypic switching differ in virulence through effects on macrophage activation.

Macrophages have a central role in the pathogenesis of cryptococcosis since they are an important line of defense, serve as a site for fungal replication, and also can contribute to tissue damage. The objective of this study was to investigate the interaction of macrophages with cells from smooth-colony variants (SM) and mucoid-colony variants (MC) arising from phenotypic switching of Cryptococcus neoformans. Alveolar macrophages (AMs) isolated from SM- and MC-infected mice exhibited differences in gene and surface expression of PD-L1, PD-L2, and major histocompatibility class II (MHC-II). PD-L1 and PD-L2 are the ligands for PD1 and are differentially regulated in Th1- and Th2-type cells. In addition, macrophage activation in SM- and MC-infected mice was characterized as alternatively activated. Flow cytometric and cytokine analysis demonstrated that MC infection was associated with the emergence of Th17 cells and higher levels of interleukin-17 (IL-17) in lung tissue, which were reduced by AM depletion. In conclusion, our results indicate that macrophages play a significant role in maintaining damage-promoting inflammation in the lung during MC infection, which ultimately results in death.

Measurement of staphylococcal enterotoxin B in serum and culture supernatant with a capture enzyme-linked immunosorbent assay.

Staphylococcal enterotoxin B (SEB) is a select agent because it is a potent mitogen that elicits life-threatening polyclonal T-cell proliferation and cytokine production at very low concentrations. Efforts are in progress to develop therapeutic reagents and vaccines that neutralize or prevent the devastating effects of this toxin. Because of its rapid binding to in vivo receptors, this toxin is difficult to detect in serum. This rapid binding also constitutes a major challenge for the development of effective therapeutic reagents that can neutralize the effects of the toxin in vivo. We have developed a highly sensitive capture enzyme-linked immunosorbent assay that detects SEB in body fluids at very low levels. With this assay, the peak levels of SEB in serum and renal clearance can be measured in mice. After either oral ingestion or nasal inhalation of SEB by mice, this assay documents the transcytosis of SEB across the mucosal membranes into serum within 2 h. Furthermore, this assay was used to compare the SEB levels in different murine models for SEB-induced lethal shock and demonstrated that the coadministration of toxin-enhancing chemicals, such as D-galactosamine and lipopolysaccharide, can alter the peak serum SEB levels. Hence, this assay is a potentially useful tool for the study of the pharmacokinetics of SEB and the effects of potential therapeutic reagents on serum SEB levels.

Lactic acidosis after treatment with linezolid.

Linezolid is currently indicated to treat vancomycin-resistant Enterococcus faecium infections, nosocomial pneumonia caused by Staphylococcus aureus or Streptococcus pneumoniae, complicated and uncomplicated skin and skin structure infections, and community-acquired pneumonia. We report a case of linezolid-induced lactic acidosis during treatment of vancomycin-resistant enterococcal bacteremia after mitral valve replacement and permanent pacemaker implantation. We also review the current literature describing other cases of linezolid-associated hyperlactatemia.

Biofilm formation by and antifungal susceptibility of Candida isolates from urine.

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC(50) of <1 mug/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.

Phagocytic efficacy of macrophage-like cells as a function of cell cycle and Fcgamma receptors (FcgammaR) and complement receptor (CR)3 expression.

Previous studies have shown that the efficiency of phagocytosis is a function of cell cycle and that phagocytosis promotes cell cycle progression. Because phagocytosis is dependent on cellular receptors we hypothesized that Fcgamma receptors (FcgammaR) and complement receptors (CR) expression varied with cell cycle. Consequently, we used centrifugal elutriation of macrophage-like cells, fluorescence activated cell sorting analysis and receptor staining to investigate expression of FcgammaR and CR as a function of cell cycle. We confirmed that FcgammaR expression on macrophage-like cells increased as the cells progressed from G1 to G2 phases. Moreover, CR3 expression varied as a function of cell cycle in a manner similar to FcgammaR. Correlation of receptor expression with cell size showed that FcgammaR and CR3 expression on macrophages was determined largely by cell size enlargement during the cell cycle. The efficacy of both Fc- and complement-mediated phagocytosis of live Cryptococcus neoformans (Cn) showed a biphasic pattern with the efficacy of phagocytosis decreasing when the cells approached the G1-S interface, which paralleled the changes in receptor surface expression when cells exited G1 phase. Live Cn cells were significantly more resistant to phagocytosis than dead cells at all stages of macrophage-like cell cycle. In contrast to live cells, the efficacy of phagocytosis of dead Cn decreased as surface receptor expression increased. Hence, the efficacy of phagocytosis in this system as function of cell cycle is not related to phagocytic receptor expression.

Phenotypic switching in a Cryptococcus neoformans variety gattii strain is associated with changes in virulence and promotes dissemination to the central nervous system.

This is the first report of a Cryptococcus neoformans var. gattii strain (serotype B) that switches reversibly between its parent mucoid (NP1-MC) colony morphology and a smooth (NP1-SM) colony morphology. Similar to C. neoformans var. grubii and C. neoformans var. neoformans strains, the switch is associated with changes in the polysaccharide capsule and virulence in animal models. In murine infection models, NP1-MC is significantly more virulent than NP1-SM (P < 0.021). In contrast to the serotype A and D strains, the serotype B strain switches in vivo reversibly between both colony morphologies. The polysaccharide of NP1-MC exhibits a thicker capsule, and thus NP1-MC exhibits enhanced intracellular survival in macrophages. Consistent with this finding, switching to the mucoid variant is observed in pulmonary infection with NP1-SM. In contrast, the thin polysaccharide capsule of NP1-SM permits better crossing of the blood-brain barrier. In this regard, only smooth colonies were grown from brain homogenates of NP1-MC-infected mice. Our findings have important implications for the pathogenesis of cryptococcosis and suggest that phenotypic switching affects host-pathogen interactions in the local microenvironment. This altered interaction then selects for specific colony variants to arise in a pathogen population.

Phenotypic switching in Cryptococcus neoformans.

Phenotypic switching has been described in serotype A and D strains of Cryptococcus neoformans. It occurs in vivo during chronic infection and is associated with differential gene expression and changes in virulence. The switch involves changes in the polysaccharide capsule and cell wall that affect the yeast's ability to resist phagocytosis. In addition, the phenotypic switch variants elicit qualitatively different inflammatory responses in the host. In animal models of chronic cryptococosis, the immune response of the host ultimately determines which of the switch variants are selected and maintained. The importance of phenotypic switching is further underscored by several findings that are relevant in the setting of human disease. These include the ability of the mucoid colony variant of RC-2 (RC-2 MC) but not the smooth variant (RC-2 SM) to promote increased intracerebral pressure in a rat model of cryptococcal meningitis. Furthermore, chemotherapeutic and immunological antifungal interventions can promote the selection of the RC-2 MC variant during chronic murine infection.

Molecular epidemiology of clinical Cryptococcus neoformans strains from India.

Little is known about the molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans in India, a country now in the midst of an epidemic of AIDS-related cryptococcosis. We studied 57 clinical isolates from several regions in India, of which 51 were C. neoformans var. grubii, 1 was C. neoformans var. neoformans, and 5 were C. neoformans var. gattii. This strain set included 18 additional sequential isolates from 14 patients. Strains were characterized phenotypically by measuring the polysaccharide capsule and by determining the MICs of standard antifungals. Molecular typing was performed by a PCR-based method using the minisatellite-specific core sequence (M13), by electrophoretic karyotyping, by restriction fragment length polymorphisms with the C. neoformans transposon 1 (TCN-1), and by URA5 DNA sequence analysis. Overall, Indian isolates were less heterogeneous than isolates from other regions and included a subset that clustered into one group based on URA5 DNA sequence analysis. In summary, our results demonstrate (i) differences in genetic diversity of C. neoformans isolates from India compared to isolates from other regions in the world; (ii) that DNA typing with the TCN-1 probe can adequately distinguish C. neoformans var. grubii strains; (iii) that TCN-1 sequences are absent in many C. neoformans var. gattii strains, supporting previous studies indicating that these strains have a limited geographical dispersal; and (iv) that human cryptococcal infection can be associated with microevolution of the infecting strain and by simultaneous coinfection with two distinct C. neoformans strains.

Phenotypic switching of Cryptococcus neoformans can produce variants that elicit increased intracranial pressure in a rat model of cryptococcal meningoencephalitis.

Increased intracranial pressure (ICP) plays an important role in the morbidity and mortality of cryptococcal meningoencephalitis. The microbial and host factors that contribute to the development of increased ICP are poorly understood. We found that phenotypic switch variants of Cryptococcus neoformans (smooth and mucoid) differed in their abilities to promote increased ICP in a rat model of cryptococcal meningitis. Rats infected with the mucoid variant developed increased ICP, whereas rats infected with the smooth parent did not. This trend correlated with a shorter survival time and a higher cerebrospinal fluid (CSF) fungal burden for mucoid variant-infected rats, although brain fungal burdens were comparable between mucoid variant- and smooth parent-infected rats. Magnetic resonance imaging revealed enhanced T2 signal intensity over the surfaces of the brains of mucoid variant-infected rats. In addition, more polysaccharide accumulated in the CSF and brains of mucoid variant-infected rats. The accumulation of glucorunoxylomannan was associated with elevated levels of MCP-1 (CCL2) and, accordingly, a more pronounced but ineffective monocytic inflammatory response in the meninges of mucoid variant-infected rats. In summary, these findings suggest that strain-specific characteristics can influence the development of increased ICP and indicate a manner in which phenotypic switching could influence the outcome of a central nervous system infection.

Phenotypic switching of Cryptococcus neoformans occurs in vivo and influences the outcome of infection.

Phenotypic switching has been linked to the virulence of many pathogens, including fungi. However, it has not been conclusively shown to occur in vivo or to influence the outcome of infection. Cryptococcus neoformans undergoes phenotypic switching in vitro to colony types that differ in their virulence in mice. In this study, we asked whether C. neoformans undergoes phenotypic switching in vivo and whether this phenomenon contributes to virulence. By using a small inoculum to preclude the introduction of variants that had already switched during in vitro propagation, we demonstrated that in vivo switching to a mucoid phenotype occurred in two mice strains and was associated with a lethal outcome. Phenotypic switching resulted in changes of the capsular polysaccharide that inhibited phagocytosis by alveolar macrophages. This promoted a more vigorous inflammatory response and rapid demise. These data document in vivo switching in a fungus and associate this phenomenon with enhanced virulence and a lethal outcome. The importance of this finding is underscored by the increased likelihood of phenotypic switching in chronic cryptococcosis; thus this mechanism may account for the inability to eradicate the organism in immunocompromised hosts.

Evidence of zoonotic transmission of Cryptococcus neoformans from a pet cockatoo to an immunocompromised patient.

BACKGROUND: Although cryptococcosis has been associated with birds for almost 50 years, point sources for infection have not been identified. OBJECTIVE: To document zoonotic transmission of Cryptococcus neoformans. DESIGN: Case report. SETTING: A home in Boston, Massachusetts. PATIENT: A 72-year-old woman who received a diagnosis of cryptococcal meningitis in November 1998. The patient, who had been taking immunosuppressant drugs since undergoing renal transplantation in 1989, owned a pet cockatoo. MEASUREMENTS: Cryptococcus neoformans was isolated from the feces of the cockatoo. Isolates from excreta and from the patient were compared by using biochemical profiles, monoclonal antibody binding patterns, restriction fragment length polymorphism analysis, and karyotyping. RESULTS: The isolates from the patient and the cockatoo had identical biochemical profiles, the same monoclonal antibody immunofluorescence patterns, and indistinguishable patterns on restriction fragment length polymorphism analysis and karyotyping. CONCLUSIONS: The indistinguishable patient and cockatoo isolates strongly suggest that the patient's infection resulted from exposure to aerosolized cockatoo excreta. Although the incidence of cryptococcal infection due to such exposure is unknown, it may be prudent to advise immunocompromised patients to avoid pet birds and avian excreta.

Fungal morphogenesis and virulence.

Phenotypic variability in pathogenic fungi has long been correlated with virulence, but specific genetic and molecular mechanisms are only recently being unraveled. Fungal morphogenesis, reflecting the expression of several regulated genes, and the capacity of the rising forms or phases to cause disease has been focused on at the XIVth Congress of the International Society for Human and Animal Mycology. Three experimental models of pathogenic fungi have been discussed. In Cryptococcus neoformans, phenotypic variability or switching represents controlled and programmed changes rather than random mutations. Evaluated phenotypic traits were the capsular polysaccharide, cell and colony morphology and virulence. In the dimorphic Paracoccidioides brasiliensis, the serine-thiol proteinase from the yeast phase cleaves the main components of the basal membrane, thus being potentially relevant in fungal dissemination. In Candida albicans, relationships between adhesion proteins and those of lymphocytes and neutrophils are related to fungal pathogenicity. Regulation of the directional growth of hyphae and its tropic responses are correlated with the invasive potential of C. albicans.

Phenotypic switching in Cryptococcus neoformans results in changes in cellular morphology and glucuronoxylomannan structure.

Cryptococcus neoformans strains exhibit variability in their capsular polysaccharide, cell morphology, karyotype, and virulence, but the relationship between these variables is poorly understood. A hypovirulent C. neoformans 24067A isolate, which usually produces smooth (SM) colony types, was found to undergo phenotypic switching and to produce wrinkled (WR) and pseudohyphal (PH) colony types at frequencies of approximately 10(-4) to 10(-5) when plated on Sabouraud agar. Cells from these colony types had large polysaccharide capsules and PH morphology, respectively. Scanning electron microscopy showed that different colony types were the result of altered cellular packing in the colony. Phenotypic switching was associated with quantitative and qualitative changes in capsular polysaccharide. Specifically, the glucuronoxylomannan (GXM) of the WR polysaccharide differed in the proportion of structural reporter groups and in increased xylose residue content linked at the 4 to 0 position. The relative virulence of the colony types was WR > PH > SM, as measured by CFU in rat lungs after intratracheal infection. Karyotype instability was observed in strain 24067A and involved primarily two chromosomes. Colonies with an alternative colony type exhibited more karyotype changes, which did not revert to the original karyotype in reverted colonies. In summary, this study revealed that phenotypic switching in C. neoformans (i) can produce WR colonies consisting of cells with either large capsule or PH morphology, (ii) is associated with production of structurally different GXM, (iii) is commonly associated with karyotype changes, (iv) can produce cells of PH morphology, and (v) can increase the virulence of a strain. Hence, phenotypic switching is an adaptive mechanism linked to virulence that can generate cell types with very different biological characteristics.

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.

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.

Genetic relationship between Cryptococcus neoformans var. neoformans strains of serotypes A and D.

Cryptococcus neoformans serotypes A and D are responsible for the overwhelming majority of infections in patients with AIDS. The genetic relationship between the serotypes is poorly understood, but there are significant differences in the epidemiology and clinical presentation of serotype A and D infections. We evaluated the genetic relationship between reference C. neoformans strains belonging to serotypes A and D by analyzing their URA5 sequences and restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element 1 (CNRE-1) probe. The results were compared to those previously obtained for isolates from Brazil and New York City by the same typing methods, and dendrograms were generated. Serotype A and D strains produced distinct RFLP patterns consistent with their separation into two major clusters in the dendrogram generated on the basis of RFLP data. Similarly, serotype A and D strains clustered independently of the basis of the nucleotide sequences of their URA5 genes. Pairwise comparisons revealed average numbers of nucleotide differences within serotypes A and D of 3.0 +/- 1.7 and 7.2 +/- 3.4, respectively (P < 0.0001), and between serotypes A and D of 41.9 +/- 2.7. In summary, our results indicate phylogenetic differences between the two serotypes of C. neoformans var. neoformans and suggest that these serotypes could probably be considered different varieties of C. neoformans.

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.

Frequency distribution of cytomegalovirus envelope glycoprotein genotypes in bone marrow transplant recipients.

Using restriction analysis of polymerase chain reaction (PCR)-amplified DNA, the cytomegalovirus (CMV) envelope glycoprotein (gB and gH) genotypes were determined for virus isolates from 128 bone marrow transplant recipients with fatal or nonfatal CMV. All isolates could be assigned to one of four gB and gH genotypes previously identified by DNA sequencing studies. Isolates of gB type 1 were more commonly found to be of gH type 2, whereas gB types 2-4 were more commonly linked to gH type 1. A small frequency of recombination with gB was detected by restriction analysis of DNA from variable regions of the gp55 and gp116 domains. Multiple isolates from various sites of 29 patients were typed and, with three exceptions, the gB genotype remained constant in all isolates from a single patient. Patients who survived CMV infection more commonly shed virus of gB type 1 than those who died (P = .003). This significant difference of gB types among patient subsets is unexplained but raises the possibility that gB genotypes may serve as a marker for pathogenicity of CMV strains in marrow transplant patients.

Declining lymphocyte counts following cytomegalovirus (CMV) infection are associated with fatal CMV disease in bone marrow transplant patients.

A previous pilot study conducted on 12 bone marrow transplant recipients suggested that detection of cytomegalovirus (CMV) in lymphocytes was associated with a drop in lymphocyte counts and death due to CMV disease. To test the association between decreasing lymphocytes and CMV-related death, we undertook a retrospective study of 332 CMV-infected patients transplanted between 1987 and 1990. The patients were divided into three groups: I = 170 patients who survived their infection and were alive at the time of the study; II = 103 patients who died of causes other than CMV infection; and III = 59 patients who died of CMV disease. Lymphocyte counts were analyzed during a 24-day period, starting 10 days before the day of first positive CMV culture (day 0). Lymphocyte counts were significantly lower in Group III from day 0 through day +14 (p < .001 vs. group I; and p = .002 vs. group II). Multivariate statistical analysis was used to adjust for other differences between the groups that might influence lymphocyte numbers. Average lymphocyte counts in patients who died of CMV disease decreased by an average of 35% after day 0. The differences in lymphocyte counts remained independent of the effects of acute graft-vs.-host disease (GVHD), time since transplant, transplant type, and high-dose steroid treatment. In summary, these data suggest that in some patients a drop in lymphocyte counts is a consequence of CMV infection associated with fatal CMV disease. Whether this can be attributed to direct infection of lymphocytes, a defective immune response, or some other mechanism remains to be determined.