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Background The shift to more transmissible but less virulent strains of SARS-CoV-2 has altered the risk calculation for infection. Particularly among young adults, the economic burden of lost work due to isolation exceeds the economic burden of morbidity due to infection. Testing strategies must adapt to these changing circumstances. Methods We modeled six testing strategies to estimate total societal costs for symptomatic people 18-49 years old: isolation of all individuals with no testing, rapid antigen test (RAg), RAg followed by a second RAg 48h later if first negative, RAg followed by a polymerase chain reaction (PCR) if negative, RAg followed by a PCR if positive, and PCR alone. We calculated costs for hypothetical cohorts of 100 symptomatic healthcare workers tested with each strategy; we included testing costs, lost wages, and hospitalization costs for the index, secondary, and tertiary cases. Key assumptions were 5% prevalence of infection, sensitivity of first/second RAg 40/80% with 97% specificity, PCR sensitivity/specificity 95/99%, all individuals isolate at symptom onset, are tested the same day, and isolate for 5 days if positive. RAg results were available the same day, PCR results were available the next day (Figure 1). One-way sensitivity analyses were performed for RAg sensitivity (20-80%) and positivity rate (1-80%). Results The least expensive strategy was RAg alone (Figure 2). This was primarily driven by its low sensitivity, which reduced lost wages at the expense of missing cases. At a threshold for RAg sensitivity lower than 29%, PCR testing alone became the cheapest strategy. When the positivity rate was > 6% confirming a negative RAg with a PCR became the cheapest strategy, closely followed by PCR alone. At a positivity rate of > 29%, isolation without testing was cheapest followed by confirming a negative RAg with a PCR and by the serial RAg test strategies (Figure 3). Conclusion In relatively young, healthy populations, a single rapid test was the least expensive strategy when the positivity rate was < 6%, testing that included PCR became cheapest at intermediate positivity, and empiric isolation was cheapest at positivity > 29%. Calibrating SARS-CoV-2 test strategies based on epidemiology may save societal costs. Disclosures Nicholas A. Turner, MD, MHSc, Aperio: Advisor/Consultant.

Background Understanding the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for public health control efforts. Social, demographic, and political characteristics at the United States (US) county level might be associated with changes in SARS-CoV-2 case incidence. Methods We conducted a retrospective analysis of the relationship between the change in reported SARS-CoV-2 case counts at the US county level during 1 June–30 June 2020 and social, demographic, and political characteristics of the county. Results Of 3142 US counties, 1023 were included in the analysis: 678 (66.3%) had increasing and 345 (33.7%) nonincreasing SARS-CoV-2 case counts between 1 June and 30 June 2020. In bivariate analysis, counties with increasing case counts had a significantly higher Social Deprivation Index (median, 48 [interquartile range {IQR}, 24–72]) than counties with nonincreasing case counts (median, 40 [IQR, 19–66]; P = .009). Counties with increasing case counts were significantly more likely to be metropolitan areas of 250 000–1 million population (P < .001), to have a higher percentage of black residents (9% vs 6%; P = .013), and to have voted for the Republican presidential candidate in 2016 by a ≥10-point margin (P = .044). In the multivariable model, metropolitan areas of 250 000–1 million population, higher percentage of black residents, and a ≥10-point Republican victory were independently associated with increasing case counts. Conclusions Increasing case counts of SARS-CoV-2 in the US during June 2020 were associated with a combination of sociodemographic and political factors. Addressing social disadvantage and differential belief systems that may correspond with political alignment will play a critical role in pandemic control.

BackgroundUnderstanding the epidemiology of SARS-CoV-2 is essential for public health control efforts. Social, demographic, and political characteristics at the US county level might be associated with the trajectories of SARS-CoV-2 case incidence.ObjectiveTo understand how underlying social, demographic, and political characteristics at the US county level might be associated with the trajectories of SARS-CoV-2 case incidence.DesignRetrospective analysis of the trajectory of reported SARS-CoV-2 case counts at the US county level during June 1, 2020 – June 30,2020 and social, demographic, and political characteristics of the county.SettingUnited States.ParticipantsReported SARS-CoV-2 cases.ExposuresMetropolitan designation, Social Deprivation Index (SDI), 2016 Republican Presidential Candidate Victory.Main Outcomes and MeasuresSARS-CoV-2 case incidence.Results1023/3142 US counties were included in the analysis. 678 (66.3%) had increasing SARS-CoV-2 case counts between June 1 – June 30, 2020. In univariate analysis, counties with increasing case counts had a significantly higher SDI (median 48, IQR 24 – 72) than counties with non-increasing case counts (median 40, IQR 19 – 66; p=0.009). In the multivariable model, metropolitan areas of 250,000 – 1 million population, higher percentage of Black residents and a 10-point or greater Republican victory were independently associated with increasing case counts.LimitationsThe data examines county-level voting patterns and does not account for individual voting behavior, subjecting this work to the potential for ecologic fallacy.ConclusionIncreasing case counts of SARS-CoV-2 in the US are likely driven by a combination of social disadvantage, social networks, and behavioral factors. Addressing social disadvantage and differential belief systems that may correspond with political alignment will be essential for pandemic control.

Treatment options for drug-resistant cytomegalovirus (CMV) are limited. Letermovir is a novel antiviral recently approved for CMV prophylaxis following hematopoietic cell transplantation, but its efficacy in other settings is unknown. We recently used letermovir for salvage treatment in four solid organ transplant recipients with ganciclovir-resistant CMV retinitis. All patients improved clinically without known adverse drug events. However, three patients failed to maintain virologic suppression, including two patients who developed genotypically confirmed resistance to letermovir while on therapy.

BACKGROUND Nocardia are uncommon pathogens that disproportionately afflict the immunocompromised host. Epidemiology and outcome data of Nocardia infections in transplant recipients are limited. METHODS We performed a retrospective chart review of all patients at Duke University Hospital with a history of solid organ transplant (SOT) or hematopoietic cell transplant (HCT) and at least one positive culture for Nocardia between 1996 and 2013. Our aim was to describe the epidemiology and outcomes of Nocardia infections in the transplanted host. RESULTS During the 18-year study period, 51 patients (14 HCT and 37 SOT recipients) had Nocardia infection. Nocardia incidence was stable during the study period in all populations except heart transplants, whose incidence declined. Infection occurred earlier in the HCT group than the SOT group (median time to diagnosis of 153 and 370 days, respectively). In both groups, the most common site involved was the lung. Outcomes were overall poor, especially in the HCT group with a cure rate of 29%. Heart transplant recipients had significantly better overall survival (P

Patients admitted with pneumonia are at higher risk for HIV and should be routinely screened. We examined a retrospective cohort of patients admitted to Duke University Health System with a primary diagnosis of pneumonia. During the study period, 6,951 persons were admitted with pneumonia. Of 6,646 patients without a known prior diagnosis of HIV, 1,010 (15%) had HIV testing during admission and 1,516 (23%) had a previously documented HIV test result. Forty-one (0.6%) patients had a positive HIV test during admission and 27 (0.4%) patients were diagnosed with HIV a median of 498 (IQR 112–982) days later, with median CD4 count of 64 (IQR 16–281) cells/mm3. HIV testing rates remain low in a population at high risk for HIV. At a minimum, we should be adhering to universal HIV screening recommendations, and certainly we should be screening those at higher risk. Opt-out HIV testing of pneumonia inpatients should be implemented.

Background Targeted testing and treatment of latent tuberculosis infection is a key element of tuberculosis (TB) elimination in the United States. In particular, foreign-born persons from TB-endemic countries are high priority for latent TB screening. Methods We used the DEDUCE interface to query the electronic medical records of all patients presenting to Duke University Health System from January 1, 2010 to November 1, 2017. Latent tuberculosis screening was identified using CPT codes for the tuberculin skin test (TST) and/or interferon gamma release assays (IGRA). Patients’ home addresses were mapped to census tracts; demographic data for these tracts were obtained from the American Community Survey. Higher-risk foreign born persons were defined as persons born in Africa, Asia, or Latin America. Results Thirty-six thousand eight hundred and twenty-five patients received 48,419 TSTs and 5,366 received 5,746 IGRAs during the study period. Excluding census tracts with fewer than 20 Health System patients (to reduce referral bias), census tracts with a higher proportion of higher risk residents had a greater proportion of Health System patients screened for latent TB (P < 0.001, figure). Health system patients residing in census tracts with greater proportions of higher risk foreign born residents were more likely to be screened with TST than with an IGRA (P < 0.001). Conclusion Latent TB screening was significantly but weakly associated with a greater proportion of higher risk foreign born persons in a given census tract, and persons residing in such tracts were more likely to be screened with TST, which is not preferred due to cross-reaction with the BCG vaccine. Focusing latent TB screening on higher risk areas and using more IGRAs will be necessary to optimize TB prevention efforts. Disclosures All authors: No reported disclosures.

The opportunistic fungal pathogen Cryptococcus neoformans is a leading cause of mortality among the human immunodeficiency virus/acquired immunodeficiency syndrome population and is known for frequently causing life-threatening relapses. To investigate the potential contribution of in-host microevolution to persistence and relapse, we have analyzed two serial isolates obtained from a patient with acquired immunodeficiency syndrome who suffered an initial and relapse episode of cryptococcal meningoencephalitis. Despite being identical by multilocus sequence typing, the isolates differ phenotypically, exhibiting changes in key virulence factors, nutrient acquisition, metabolic profiles, and the ability to disseminate in an animal model. Whole-genome sequencing uncovered a clonal relationship, with only a few unique differences. Of these, two key changes are expected to explain the phenotypic differences observed in the relapse isolate: loss of a predicted AT-rich interaction domain protein and changes in copy number of the left and right arms of chromosome 12. Gene deletion of the predicted transcriptional regulator produced changes in melanin, capsule, carbon source use, and dissemination in the host, consistent with the phenotype of the relapse isolate. In addition, the deletion mutant displayed altered virulence in the murine model. The observed differences suggest the relapse isolate evolved subsequent to penetration of the central nervous system and may have gained dominance following the administration of antifungal therapy. These data reveal the first molecular insights into how the Cryptococcus neoformans genome changes during infection of humans and the manner in which microevolution progresses in this deadly fungal pathogen.

Nocardia is a genus of pathogens that most commonly afflict immunocompromised hosts but may be an emerging infection among persons with bronchiectasis.To examine the epidemiology and clinical presentation of adult patients with Nocardia and bronchiectasis relative to other patient groups.We examined a retrospectively assembled cohort of adults at Duke University Hospital in Durham, North Carolina with at least one positive culture from a bodily fluid or tissue specimen for Nocardia between January 1996 and December 2013. Denominator data for key populations (e.g., bronchiectasis, transplant) were obtained using International Classification of Diseases, Ninth Revision codes. In addition, we performed a case-control analysis to examine the relationship between inhaled corticosteroid use and Nocardia lung infection among otherwise immunocompetent patients with bronchiectasis.We identified 183 patients with one or more cultures positive for Nocardia: 44 from 1996 to 2001, 64 from 2002 to 2007, and 75 from 2008 to 2013. Immune compromise was common (56%), particularly solid organ or hematopoietic cell transplant (30%). Infection usually was confined to the lungs (62%), followed by skin (10%), other sites (6%), brain (2%), and multiple sites (17%). Non-cystic fibrosis bronchiectasis was common among both immunocompetent (38%) and immunocompromised (10%) patients. Nocardia incidence in patients with bronchiectasis increased significantly over time, but there was no significant change in Nocardia incidence in hematopoietic cell or solid organ transplant recipients (our largest immunocompromised population). Among patients with bronchiectasis, Nocardia was positively but nonsignificantly associated with use of inhaled corticosteroids (odds ratio, 1.8; 95% confidence interval, 0.7-4.4).The increasing incidence of Nocardia infections at our medical center appears to be driven by increased incidence in patients with bronchiectasis rather than increases in immunocompromised populations. It is unclear whether increased environmental exposures, microbiologic surveillance, or other factors account for the increased incidence of Nocardia in our patients with bronchiectasis.

Background Pedicure-associated nontuberculous mycobacterial furunculosis has been reported in the setting of either outbreaks or sporadic case reports. The epidemiology of these infections is not well understood. Methods Systematic surveillance for pedicure-associated nontuberculous mycobacterial furunculosis was conducted in 2 North Carolina counties from 1 January 2005 through 31 December 2008. A subset of implicated nail salons and control salons was inspected and sampled for nontuberculous mycobacteria. Results Forty cases of suspected or confirmed pedicure-associated nontuberculous mycobacterial furunculosis were reported during the 4-year study period. Furunculosis incidence in the surveillance region was 1.00, 0.96, 0.83, and 0.89 cases per 100,000 population in 2005, 2006, 2007, and 2008, respectively. The responsible organisms primarily belonged to the Mycobacterium chelonae/abscessus group (30 [91%] of 33 isolates). Thirteen implicated salons and 11 control salons were visited and environmentally sampled. An assortment of nontuberculous mycobacteria was cultured from footbaths, but there was no association between the species distribution of the environmental isolates and implication of the salon in human infection. Evidence of suboptimal cleaning (visible debris or surface biofilms) was observed in at least 1 footbath for 11 of 13 implicated salons and 4 of 11 control salons (P = .032). Conclusions Pedicure-associated mycobacterial furunculosis was endemic in these 2 North Carolina counties during 2005-2008. Suboptimal footbath cleaning may have contributed to these infections, which suggests straightforward means of potential prevention. The relative rarity of this type of infection in the setting of nearly ubiquitous exposure to these pathogens suggests that as yet undefined host-specific or procedure-related factors may be involved in susceptibility to these infections.

Knowing one's HIV status is particularly important in the setting of recent tuberculosis (TB) exposure. Blood tests for assessment of tuberculosis infection, such as the QuantiFERON Gold in-tube test (QFT; Cellestis Limited, Carnegie, Victoria, Australia), offer the possibility of simultaneous screening for TB and HIV with a single blood draw. We performed a cross-sectional analysis of all contacts to a highly infectious TB case in a large meatpacking factory. Twenty-two percent were foreign-born and 73% were black. Contacts were tested with both tuberculin skin testing (TST) and QFT. HIV testing was offered on an opt-out basis. Persons with TSTor=10 mm, positive QFT, and/or positive HIV test were offered latent TB treatment. Three hundred twenty-six contacts were screened: TST results were available for 266 people and an additional 24 reported a prior positive TST for a total of 290 persons with any TST result (89.0%). Adequate QFT specimens were obtained for 312 (95.7%) of persons. Thirty-two persons had QFT results but did not return for TST reading. Twenty-two percent met the criteria for latent TB infection. Eighty-eight percent accepted HIV testing. Two (0.7%) were HIV seropositive; both individuals were already aware of their HIV status, but one had stopped care a year previously. None of the HIV-seropositive persons had latent TB, but all were offered latent TB treatment per standard guidelines. This demonstrates that opt-out HIV testing combined with QFT in a large TB contact investigation was feasible and useful. HIV testing was also widely accepted. Pairing QFT with opt-out HIV testing should be strongly considered when possible.

We evaluated cell-mediated immune (CMI) responses in mice given a pulmonary infection with aCryptococcus neoformansstrain engineered to produce the Th1-type cytokine gamma interferon (IFN-γ). Mice given a pulmonary infection with an IFN-γ-producingC. neoformansstrain were able to resolve the primary infection and demonstrated complete (100%) protection against a second pulmonary challenge with a pathogenicC. neoformansstrain. Pulmonary cytokine analyses showed that Th1-type/proinflammatory cytokine and chemokine expression were significantly higher and Th2-type cytokine expression was significantly lower in mice infected with the IFN-γ-producingC. neoformansstrain compared to wild-type-infected mice. This increased pulmonary Th1-type cytokine expression was also associated with significantly lower pulmonary fungal burden and significantly higher pulmonary leukocyte and T-lymphocyte recruitment in mice infected with the IFN-γ-producingC. neoformansstrain compared to wild-type-infected mice. Our results demonstrate that pulmonary infection of mice with aC. neoformansstrain expressing IFN-γ results in the stimulation of local Th1-type anti-cryptococcal CMI responses and the development of protective host immunity against future pulmonary cryptococcal infections. The use of fungi engineered to produce host cytokines is a novel method to study immune responses to infection and may be useful in developing vaccine strategies in humans.

The ability of Cryptococcus neoformans to grow at the mammalian body temperature (37°C to 39°C) is a well-established virulence factor. Growth of C. neoformans at this physiological temperature requires calcineurin, a Ca 2+ /calmodulin-dependent protein phosphatase. When cytosolic calcium concentrations are low (∼50 to 100 nM), calcineurin is inactive and becomes active only when cytosolic calcium concentrations rise (∼1 to 10 μM) through the activation of calcium channels. In this study we analyzed the function of Cch1 in C. neoformans and found that Cch1 is a Ca 2+ -permeable channel that mediates calcium entry in C. neoformans . Analysis of the Cch1 protein sequence revealed differences in the voltage sensor (S4 regions), suggesting that Cch1 may have diminished voltage sensitivity or possibly an alternative gating mechanism. The inability of the cch1 mutant to grow under conditions of limited extracellular calcium concentrations ([Ca 2+ ] extracellular , ∼100 nM) suggested that Cch1 was required for calcium uptake in low-calcium environments. These results are consistent with the role of ScCch1 in mediating high-affinity calcium uptake in Saccharomyces cerevisiae . Although the growth defect of the cch1 mutant under conditions of limited [Ca 2+ ] extracellular (∼100 nM) became more severe with increasing temperature (25°C to 38.5°), this temperature sensitivity was not observed when the cch1 mutant was grown on rich medium ([Ca 2+ ] extracellular , ∼0.140 mM). Accordingly, the cch1 mutant strain displayed only attenuated virulence when tested in the mouse inhalation model of cryptococcosis, further suggesting that C. neoformans may have a limited requirement for Cch1 and that this requirement appears to include ion stress tolerance.

The Gα protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein GprH and also shares structural similarity with the Saccharomyces cerevisiae receptor Gpr1. gpr4 mutants exhibited reduced capsule production and mating defects, similar to gpa1 mutants, and exogenous cAMP suppressed both gpr4 mutant phenotypes. Epistasis analysis provides further evidence that Gpr4 functions upstream of the Gα subunit Gpa1. Gpr4-Gpr4 homomeric interactions were observed in the yeast two-hybrid assay, and Gpr4 was shown to physically interact with Gpa1 in the split-ubiquitin system. A Gpr4::DsRED fusion protein was localized to the plasma membrane and methionine was found to trigger receptor internalization. The analysis of intracellular cAMP levels showed that gpr4 mutants still respond to glucose but not to certain amino acids, such as methionine. Amino acids might serve as ligands for Gpr4 and could contribute to engage the cAMP-PKA pathway. Activation of the cAMP-PKA pathway by glucose and amino acids represents a nutrient coincidence detection system shared in other pathogenic fungi.

Cryptococcus neoformans is an encapsulated fungal pathogen that primarily infects the central nervous system of immunocompromised individuals, causing life-threatening meningoencephalitis. The capacity of C. neoformans to subvert host defenses and disseminate by intracellular parasitism of alveolar macrophages in the immune-compromised host has led to studies to evaluate genes associated with C. neoformans resistance to oxidative stress. In the present study, we identify and characterize a C. neoformans homologue to SKN7 , a transcription factor in Saccharomyces cerevisiae that regulates the oxidative stress response, cell cycle, and cell wall biosynthesis. To examine the contribution of SKN7 in the pathogenesis of fungal infections, we created skn7 mutants via targeted disruption. The skn7 mutants were observed to be more susceptible to reactive oxygen species in vitro and were significantly less virulent than the wild-type strain and a reconstituted strain as measured by cumulative survival in the mouse inhalational model. The Skn7 protein was observed to be important for expression of thioredoxin reductase in response to oxidative challenge. Interestingly, skn7 mutants were also observed to flocculate following in vitro culture, a novel phenotype not observed in skn7 mutants derived from other fungi. These findings demonstrate that SKN7 contributes to the virulence composite but is not required for pathogenicity in C. neoformans . In addition, flocculation of C. neoformans skn7 mutants suggests a potentially unique function of SKN7 not previously observed in other cryptococcal strains or skn7 mutants.

We conducted a study to determine the antifungal susceptibility of vaginal Candida isolates from HIV-infected Brazilian women. Among 127 women enrolled, positive cultures for yeast were obtained from 31 of 38 (81%) women with symptomatic vulvovaginitis, and from 41 of 89 (46%) asymptomatic women. Susceptibility testing demonstrated 11 of the 72 isolates had either resistance or dose-dependent susceptibility to azole drugs, including four Candida albicans strains. Expression of the MDR1, CDR1, CDR2 and ERG11 genes was evaluated in all of the C. albicans isolates, and all four of the strains with reduced susceptibility to fluconazole had increased expression of CDR1 as compared to the fluconazole-sensitive strains. No increased expression of the other genes was identified. This large survey of Candida isolates from HIV-infected women from Brazil demonstrates that reduced susceptibility to azoles occurs at a low frequency among vaginal yeast isolates, and when present in C. albicans, azole resistance is associated with increased expression of CDR1.

Cryptococcus neoformans is an opportunistic pathogen and the leading cause of fungal meningitis. To survive within the host, this organism must be able to protect itself from oxidative stress. Cytochrome c peroxidase (Ccp1) is a mitochondrial antioxidant that catalyzes the degradation of hydrogen peroxide. In the present study, we characterized the contribution of the C. neoformans Ccp1 to antioxidant defense and for virulence. Consistent with studies of Ccp1 function in Sacchromyces cerevisiae, we found that Ccp1 contributes to resistance against exogenous oxidative stress in vitro. However, the oxidative stress phenotype does not diminish the virulence of ccp1 mutant strains in a murine model of C. neoformans disease. These results suggest that Ccp1 is involved in a complex system of protection against exogenous oxidative stress and that the elimination of this component of the antioxidant defense system does not diminish the virulence of C. neoformans.

The impact of syphilis reverse sequence screening has not been evaluated in community outreach. Using reverse sequence screening in neighborhoods identified with geographic information systems, we found that among 239 participants, 45 (19%) were seropositive. Of these, 3 (7%) had untreated syphilis, 33 (73%) had previously treated syphilis infection, and 9 (20%) had negative nontreponemal test results.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

Cryptococcus neoformans serotype A strains commonly infect immunocompromised patients to cause fungal meningitis. To understand the basis of serotype A cryptococcal infections in apparently immunocompetent patients, we tested two hypotheses: the strains were naturally occurring hypervirulent pkr1 (PKA regulatory subunit) mutants, or the strains were hybrids with C. neoformans var. gattii strains that normally infect immunocompetent individuals. Analysis of clinical isolates obtained from apparently immunocompetent individuals from three continents revealed that none were pkr1 mutants, but several exhibited phenotypes consistent with perturbations in cAMP signaling. Additionally, none of the strains were unusual hybrids with gattii strains. Except for one strain that was an AD hybrid, all others were serotype A (var. grubii) isolates. Taken together, our findings indicate that the ability of these clinical isolates to infect apparently normal individuals may be attributable to mutations other than pkr1 and/or underlying immune system impairment in patients.

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO [1–6]. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity [7–11]. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS −/− animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.

Cryptococcus neoformans is a fungal pathogen that, after inhalation, can disseminate to the brain. Host alveolar macrophages (AMs) represent the first defense against the fungus. Once phagocytosed by AMs, fungal cells are killed by a concerted mechanism, involving the host-cellular response. If the cellular response is impaired, phagocytosis of the fungus may be detrimental for the host, since C. neoformans can grow within macrophages. Here, we identified a novel cryptococcal gene encoding antiphagocytic protein 1 (App1). App1 is a cryptococcal cytoplasmic protein that is secreted extracellularly and found in the serum of infected patients. App1 does not affect melanin production, capsule formation, or growth of C. neoformans. Treatment with recombinant App1 inhibited phagocytosis of fungal cells through a complement-mediated mechanism, and Deltaapp1 mutant is readily phagocytosed by AMs. Interestingly, the Deltaapp1 mutant strain showed a decreased virulence in mice deficient for complement C5 (A/Jcr), but it was hypervirulent in mice deficient for T and NK cells (Tgepsilon26). This study identifies App1 as a novel regulator of virulence for C. neoformans, and it highlights that internalization of fungal cells by AMs increases the dissemination of C. neoformans when the host cellular response is impaired.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised patients. The Ca 2+ -calmodulin-activated protein phosphatase calcineurin is necessary for virulence of C. neoformans . Mutants lacking the calcineurin catalytic (Cna1) or regulatory (Cnb1) subunit fail to grow at elevated temperature and are defective in virulence and hyphal elongation. Here we isolated a multicopy suppressor gene, CTS1 , which restores growth of a calcineurin mutant strain at 37°C. The CTS1 gene (for calcineurin temperature suppressor 1) encodes a protein containing a C2 domain and a leucine zipper motif that may function as an effector of calcineurin. The CTS1 gene was disrupted by homologous recombination, and cts1 mutants were viable but exhibited defects in cell separation, growth, mating, and haploid fruiting. In addition, cts1 mutants were inviable when calcineurin was mutated or inhibited. Taken together, these findings suggest that calcineurin and Cts1 function in parallel pathways that regulate growth, cell separation, and hyphal elongation.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle in which the alpha allele of the mating type locus is linked to virulence and haploid differentiation. Here we analysed a conserved MAP kinase cascade composed of mating-type specific (Ste11alpha, Ste12alpha) and non-specific (Ste7, Cpk1) elements. Gene disruption experiments demonstrate that this specialized MAP kinase pathway is required for both mating and cell type-specific differentiation but not for virulence. The Ste11alpha, Ste7 and Cpk1 kinases were found to act as a co-ordinate signalling module, whereas the Ste12alpha transcription factor functions with a redundant partner or in a branched or parallel signalling pathway. Our studies illustrate how MAP kinase cascades can be constructed from cell type-specific and non-specific components, yielding pathways that contribute to cell type-specific patterns of signalling and differentiation.

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37 degrees C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a beta-1,3-glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeastCryptococcus neoformansand made mutants via targeted disruption. Thesod1mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. Asod1mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that thesod1strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity inC. neoformans; (ii) the decreased virulence of thesod1strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems inC. neoformanscan compensate for the loss of the Cu,Zn SOD in vivo.

Functional genomics has become a major focus in the study of microbial pathogenesis. This study used a functional genomic tool, differential display reverse transcription-PCR, to identify a transcriptional profile of Cryptococcus neoformans cells as they produced meningitis in an immunosuppressed host. This serial global gene expression during infection allowed for the identification of up- and down-regulated genes during infection. During this profiling, a single gene for the enzyme isocitrate lyase (ICL1) was found to be up regulated at 1 week of infection in a rabbit meningitis model and during a time of maximum host cellular response. The finding suggested that this enzyme and the glyoxylate shunt pathway are important to this yeast's energy production during infection. However, site-directed icl1 mutants had no apparent virulence defect in two animal models and no growth defect within macrophages. These observations suggest that although the yeast responded to a certain environmental cue(s) by an increase in ICL1 expression during infection, this gene was not necessary for progression of a C. neoformans infection. Compounds that specifically target only ICL1 are unlikely to cripple C. neoformans growth in vivo.

The Cryptococcus neoformans MET3 cDNA (encoding ATP sulfurylase) was cloned by complementation of the corresponding met3 mutation in Saccharomyces cerevisiae. Sequence analysis showed high similarity between the deduced amino acid sequence of the C. neoformans Met3p and other fungal ATP sulfurylases. A C. neoformans met3 mutant was made by targeted insertional mutagenesis, which had the expected auxotrophic phenotype, and reconstituted the met3 mutant to Met+. In vitro, the C. neoformans met3 mutant had a substantial defect in melanin formation, significantly reduced growth rate, and greatly increased thermotolerance. In the murine inhalation infection model, the met3 mutant was avirulent and was deficient in its ability to survive in mice. It is concluded that, in contrast to the yeast form of Histoplasma capsulatum, in C. neoformans the sulfate-assimilation arm of the methionine biosynthetic pathway plays an important role in vitro, even in the presence of abundant exogenous methionine, and is critical for virulence, and indeed for survival, in vivo.

Cryptococcus neoformans is a pathogenic fungus with a defined sexual cycle involving haploid MAT α and MAT a cells. Interestingly, MAT α strains are more common, are more virulent than congenic MAT a strains, and undergo haploid fruiting in response to nitrogen limitation or MAT a cells. Three genes encoding the MFα pheromone were identified in the MAT α mating-type locus and shown to be transcriptionally induced by limiting nutrients and coculture with MAT a cells. The MFα1 , MFα2 , and MFα3 genes were mutated, individually and in combination. MAT α strains lacking MFα pheromone failed to induce morphological changes in MAT a cells. Pheromoneless MAT α mutants were fusion and mating impaired but not sterile and mated at ∼1% the wild-type level. The pheromoneless MAT α mutants were also partially defective in haploid fruiting, and overexpression of MFα pheromone enhanced haploid fruiting. Overexpression of MFa pheromone also enhanced haploid fruiting of MAT α cells and stimulated conjugation tube formation in MAT a cells. A conserved G-protein activated mitogen-activated protein kinase signaling pathway was found to be required for both induction and response to mating pheromones. The MFα pheromone was not essential for virulence of C. neoformans but does contribute to the overall virulence composite. These studies define paracrine and autocrine pheromone response pathways that signal mating and differentiation of this pathogenic fungus.

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.

The Ras1 signal transduction pathway controls the ability of the pathogenic fungus Cryptococcus neoformans to grow at high temperatures and to mate. A second RAS gene was identified in this organism. RAS2 is expressed at a very low level compared to RAS1, and a ras2 mutation caused no alterations in vegetative growth rate, differentiation or virulence factor expression. The ras2 mutant strain was equally virulent to the wild-type strain in the murine inhalational model of cryptococcosis. Although a ras1 ras2 double mutant strain is viable, mutation of both RAS genes results in a decreased growth rate at all temperatures compared to strains with either single mutation. Overexpression of the RAS2 gene completely suppressed the ras1 mutant mating defect and partially suppressed its high temperature growth defect. After prolonged incubation at a restrictive temperature, the ras1 mutant demonstrated actin polarity defects that were also partially suppressed by RAS2 overexpression. These studies indicate that the C. neoformans Ras1 and Ras2 proteins share overlapping functions, but also play distinct signalling roles. Our findings also suggest a mechanism by which Ras1 controls growth of this pathogenic fungus at 37 °C, supporting a conserved role for Ras homologues in microbial cellular differentiation, morphogenesis and virulence.

We welcome this further discussion of our results on the age of the Great Gallery rock art in the Canyonlands of Utah. The comment by Simon and Reed (1) focuses on just one of the three components of our study (2), which is presented in greater technical detail in ref. 3 and is surely our best-constrained and least-surprising result: the dating of a rock-fall that removed some of the art and thus provides a minimum age. Simon and Reed (1) point out that the Great Gallery panel is not pristine and relate the sordid human history of visitation and possible disturbance to the site. Indeed, being aware of this during our research, one of our initial hypotheses was that the rock fall may be historic. Despite the possibility of recent disturbance to some of the talus boulders, our results document that the rock fall occurred ∼900 y ago, and for the boulder we sampled a scenario of historic disturbance and exposure such as postulated by Simon and Reed (1) can be ruled out.

We assessed the clinical utility of indium 111–labeled white blood cell scans at our tertiary referral center from 2005 to 2011. Overall, scans meaningfully impacted clinical care, Background We sought to characterize the clinical utility of indium 111 (111In)–labeled white blood cell (WBC) scans by indication, to identify patient populations who might benefit most from this imaging modality. Methods Medical records for all patients who underwent 111In-labeled WBC scans at our tertiary referral center from 2005 to 2011 were reviewed. Scan indication, results, and final diagnosis were assessed independently by 2 infectious disease physicians. Reviewers also categorized the clinical utility of each scan as helpful vs not helpful with diagnosis and/or management according to prespecified criteria. Cases for which clinical utility could not be determined were excluded from the utility assessment. Results One hundred thirty-seven scans were included in this analysis; clinical utility could be determined in 132 (96%) cases. The annual number of scans decreased throughout the study period, from 26 in 2005 to 13 in 2011. Forty-one (30%) scans were positive, and 85 (62%) patients were ultimately determined to have an infection. Of the evaluable scans, 63 (48%) scans were deemed clinically useful. Clinical utility varied by scan indication: 111In-labeled WBC scans were more helpful for indications of osteomyelitis (35/50, 70% useful) or vascular access infection (10/15, 67% useful), and less helpful for evaluation of fever of unknown origin (12/35, 34% useful). Conclusions 111In-labeled WBC scans were useful for patient care less than half of the time at our center. Targeted ordering of these scans for indications in which they have greater utility, such as suspected osteomyelitis and vascular access infections, may optimize test utilization.

Infection with HIV is the most common predisposing condition for developing cryptococcosis. This chapter focuses on aspects that are specific to the coinfection of HIV and Cryptococcus neoformans. These include clinical presentation, treatment regimens, alterations of the host immune response, and concerns about drug toxicities and interactions. Soon after the HIV pandemic was recognized in the United States and Europe, it became clear that cryptococcosis was an important opportunistic pathogen in patients with AIDS. C. neoformans infection in AIDS patients can either be the result of a newly acquired primary infection or alternatively constitute a reactivation of latent C. neoformans infection. Patients with AIDS who respond to highly active antiretroviral therapy (HAART) show a greatly reduced incidence of opportunistic infections such as cryptococcosis. Experimental evidence suggests that C. neoformans coinfection can affect HIV replication. Another factor that may affect HIV-infected patients’ ability to mount a successful antifungal host response is the common abuse of methamphetamine. This drug exposure facilitates intracellular replication and inhibits intracellular killing of C. neoformans and thus affects pathogenesis in these patients. The most common manifestation of cryptococcosis in patients with HIV infection is meningoencephalitis, and central nervous system (CNS) involvement is found in the vast majority of patients with AIDS in whom cryptococcal infection is diagnosed. The treatment of AIDS-associated cryptococcal meningitis is usually divided into three stages: induction, consolidation, and maintenance.

Calcineurin is a Ca2+–calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37°C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12–FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12–FK506–calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

The basidiomycete Cryptococcus neoformans is an opportunistic human fungal pathogen that causes cryptococcal meningitis predominantly in immunocompromised patients. A well-defined sexual life cycle (20–22, 25) and the establishment of various molecular biological tools make this organism an excellent model system for studies of fungal pathogenicity (8). The life cycle of C. neoformans is characterized by a dimorphic transition between a haploid yeast form and a dikaryotic filamentous form (1). After a haploid cell senses a cell of opposite mating type (MATa or MATα), the cells produce filament-like structures called conjugation tubes that protrude toward the mating partner. After cell fusion, a dikaryotic mycelium is generated, composed of filaments characterized by unfused parental nuclei and fused clamp connections. Under proper environmental conditions, the tips of these filaments develop into swollen cells termed basidia. Within this new structure, karyogamy occurs followed by meiosis to generate four recombinant haploid nuclei. The haploid nuclei divide mitotically and bud off from the basidium to generate four long chains of basidiospores. These spores germinate and produce vegetative, yeast cells. The diploid phase of C. neoformans is normally transient, and therefore most environmental and clinical isolates are haploid. Nevertheless, rare diploid isolates have been reported based on assays determining cellular DNA contents (33–35), the analysis of randomly amplified polymorphic DNA markers (4, 37), or isozyme analysis (5). Furthermore, we and others isolated diploid strains following defined genetic crosses (32, 38, 39). Interestingly, most of the environmental and clinical isolates that are thought to be diploid belong to the unusual class of serotype AD strains (5, 34). C. neoformans has been classified into three varieties based in part on serological differences in capsular antigens. Serotype A and D strains each belong to a separate variety (varieties grubii and neoformans, respectively), whereas serotype B and C strains are classified as a single variety (variety gattii) (16, 23). A fifth, unusual serotype, AD, has also been described but is much less common than the four predominant serotypes. DNA sequence analysis has revealed that serotype B and C strains are phylogenetically much more closely related to each other than to A or D strains, which in turn are estimated to have diverged from each other approximately 18 million years ago (13, 42). Karyotype analysis revealed that the average chromosome number for C. neoformans is between 12 and 13 (28, 29, 41). The smallest chromosomes in variety gattii are 400 to 700 kb in size, whereas the smallest chromosomes of varieties neoformans and grubii are approximately 770 kb. Nucleotide sequence comparison of the URA5 gene revealed ∼8% sequence divergence between variety gattii and variety neoformans or grubii (7, 14) and ∼5% sequence divergence between varieties neoformans and grubii. Here we have characterized strains of the unusual serotype AD class to establish their origin. By fluorescence-activated cell sorter (FACS) analysis we found that serotype AD strains are diploid or aneuploid (>1n but ≤2n). Second, by PCR analysis we showed that these strains are heterozygous for serotype A- and D-specific alleles and the MATa and MATα mating-type loci. Third, we found that three serotype AD strains were self-fertile and produced filaments, basidia, and basidiospores that germinated poorly (∼5%) to produce progeny that were still diploid or aneuploid. Our findings reveal that serotype AD strains are hybrids produced by crosses between serotype A and D parental strains and suggest that evolutionary divergence and sequence differences prevent proper chromosome segregation during meiosis in AD hybrid strains.

Cryptococcus neoformans is an excellent model system for studies on the molecular pathogenesis of fungal infections. There is only one dominant selectable market that can be used in the transformation of this organism, and we wanted to develop another. We found that various strains of C. neoformans are very sensitive to the aminoglycoside antibiotic nourseothricin, and that spontaneous resistance to this drug must be an extremely rare event. Resistance to nourseothricin is conferred by the product of the nourseothricin acetyltransferase gene (nat1) from Streptomyces noursei. In order to express this gene in C. neoformans, we created a fusion construct by driving expression of natl with the promoter sequence from a C. neoformans actin gene. Biolistic transformation of the serotype A C. neoformans strain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1,000 transformants microg(-1) of DNA and 20 transformants microg(-1) of DNA, respectively. Southern blots were performed using DNA from some of the H99 transformants, and this confirmed that all of the resistant isolates had the construct integrated in a random fashion within the genome. There was no cross-resistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker in C. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.

Strategies to Identify Virulence Genes There are two basic pathways for the identification of fungal virulence genes. First, there is the direct study of genes controlling known virulence phenotypes and the pathways that control them. This type of strategy is generally associated with a known phenotype, such as adherence, capsule production, melanin synthesis, hyphal formation, or a known biochemical pathway. Previous studies help to direct this work, and there is usually a model in other organisms to follow. This strategy has been used very effectively to identify certain signal transduction pathways that control virulence genes. Model organisms have helped to establish a “road map” to follow. For instance, Saccharomyces cerevisiae can be used to identify phenotypes and genotypes for virulence studies (46), but the more virulent pathogens appear to use the genetic network differently. These are the variations that we need to discover and follow. For example, the use of CNAI (calcineurin) for high temperature growth of C. neoformans could not have been predicted by work with S. cerevisiae (35). The knowledge that GPAI, which encodes for an alpha subunit of a G-protein, is associated with the virulence of C. neoformans could not have been deduced without directly studying the pathogen (47). In fact, this signal protein and its pathway actually influence the virulence composite through its control of several virulence phenotypes (capsule, melanin, and mating). It is clear that further work on signaling pathways will be very enlightening as we attempt to dissect and understand the network of virulence genes. It is clear, also, that with a single gene disruption, these signaling genes can produce profound effects on the ability of the null mutant to produce disease. Much more work on the “signal” genes remains in order to identify (i) receptors (upstream); (ii) effecters (downstream); and (iii) the various connections (networking) to other pathways. By understanding these circuits and their regulation, it is reasonable to predict that controls on the virulence phenotype can be designed.

Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria. Cryptococcus neoformans is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy C. neoformans urease gene ( URE1 ) and used this to disrupt the native URE1 in the serotype A strain H99. The ure1 mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a ure1 mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a ure1 strain lived longer than mice infected with H99 in both models. The ure1 strain was restored to urease positivity by complementation with URE1 , and two resulting transformants were significantly more pathogenic than the ure1 strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.

The calcineurin gene was cloned and disrupted in serotype D strains of Cryptococcus neoformans . Serotype A and serotype D calcineurin mutants were inviable at 37°C and avirulent in mice, whereas only serotype A mutants were cation stress sensitive. Thus, calcineurin plays conserved and divergent roles in serotype A and serotype D strains.

The pathophysiology of oropharyngeal candidiasis in patients infected with human immunodeficiency virus (HIV) type 1 is poorly understood. Association between oropharyngeal yeast carriage and various clinical factors in HIV-1-infected patients was studied in 83 patients with no clinical evidence of thrush and no recent antifungal use. Of the clinical factors measured, the only correlate of yeast colonization was with plasma HIV-1 RNA levels (P=.001), whereas the correlation with CD4 cell count was poor (P=.36). By multivariable regression modeling, plasma HIV-1 RNA was the only parameter that correlated with the extent of colonization with Candida infection (P=.003). These data indicate that the presence and amount of asymptomatic oropharyngeal yeast carriage in persons with HIV-1 infection is more significantly correlated with plasma HIV-1 RNA levels than with CD4 cell count. Further studies on the effect of HIV-1 on oropharyngeal yeast colonization, infection, and local immunity are warranted.