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Cryptococcosis by Cryptococcus gattii is endemic in Colombia, affecting mostly immunocompetent hosts. Since antifungal susceptibility differs between molecular types of cryptococcal isolates, as reported elsewhere, the aim of this study was to determine if 42 Colombian clinical isolates, VGI, VGII and VGIII, differ in the susceptibility to commonly used antifungals, using Sensititre plates. Among the molecular types, six non-wild type isolates to fluconazole, voriconazole, and 5-flucytosine, were identified. Besides, VGI and VGII were less susceptible to 5-flucytosine and azoles, respectively, than other molecular types. These findings support the applicability of practicing susceptibility testing, which could better guide treatment in cryptococcosis. Lay Summary Cryptococcosis gattii affects immunocompetent people. For a correct treatment, antifungal susceptibility testing is essential. This study shows differences in the susceptibility to commonly used antimycotics among genotypes of Colombian clinical C. gattii isolates, some of which are non-wild-type.

Background Mortality from cryptoccocal meningitis remains high. The ACTA trial demonstrated that, compared with 2 weeks of amphotericin B (AmB) plus flucystosine (5FC), 1 week of AmB and 5FC was associated with lower mortality and 2 weeks of oral flucanozole (FLU) plus 5FC was non-inferior. Here, we assess the cost-effectiveness of these different treatment courses. Methods Participants were randomized in a ratio of 2:1:1:1:1 to 2 weeks of oral 5FC and FLU, 1 week of AmB and FLU, 1 week of AmB and 5FC, 2 weeks of AmB and FLU, or 2 weeks of AmB and 5FC in Malawi, Zambia, Cameroon, and Tanzania. Data on individual resource use and health outcomes were collected. Cost-effectiveness was measured as incremental costs per life-year saved, and non-parametric bootstrapping was done. Results Total costs per patient were US $1442 for 2 weeks of oral FLU and 5FC, $1763 for 1 week of AmB and FLU, $1861 for 1 week of AmB and 5FC, $2125 for 2 weeks of AmB and FLU, and $2285 for 2 weeks of AmB and 5FC. Compared to 2 weeks of AmB and 5FC, 1 week of AmB and 5FC was less costly and more effective and 2 weeks of oral FLU and 5FC was less costly and as effective. The incremental cost-effectiveness ratio for 1 week of AmB and 5FC versus oral FLU and 5FC was US $208 (95% confidence interval $91–1210) per life-year saved. Clinical Trials Registration ISRCTN45035509. Conclusions Both 1 week of AmB and 5FC and 2 weeks of Oral FLU and 5FC are cost-effective treatments., Compared to 2 weeks of amphotericin B (AmB) and flucystosine (5FC), both 1 week of AmB/5FC and 2 weeks of oral fluconazole (FLU) and 5FC save costs; 1 week of AmB/5FC reduces mortality rates more and is somewhat more costly.

Background Mortality from cryptococcal meningitis remains very high in Africa. In the Advancing Cryptococcal Meningitis Treatment for Africa (ACTA) trial, 2 weeks of fluconazole (FLU) plus flucytosine (5FC) was as effective and less costly than 2 weeks of amphotericin-based regimens. However, many African settings treat with FLU monotherapy, and the cost-effectiveness of adding 5FC to FLU is uncertain. Methods The effectiveness and costs of FLU+5FC were taken from ACTA, which included a costing analysis at the Zambian site. The effectiveness of FLU was derived from cohorts of consecutively enrolled patients, managed in respects other than drug therapy, as were participants in ACTA. FLU costs were derived from costs of FLU+5FC in ACTA, by subtracting 5FC drug and monitoring costs. The cost-effectiveness of FLU+5FC vs FLU alone was measured as the incremental cost-effectiveness ratio (ICER). A probabilistic sensitivity analysis assessed uncertainties and a bivariate deterministic sensitivity analysis examined the impact of varying mortality and 5FC drug costs on the ICER. Results The mean costs per patient were US $847 (95% confidence interval [CI] $776–927) for FLU+5FC, and US $628 (95% CI $557–709) for FLU. The 10-week mortality rate was 35.1% (95% CI 28.9–41.7%) with FLU+5FC and 53.8% (95% CI 43.1–64.1%) with FLU. At the current 5FC price of US $1.30 per 500 mg tablet, the ICER of 5FC+FLU versus FLU alone was US $65 (95% CI $28–208) per life-year saved. Reducing the 5FC cost to between US $0.80 and US $0.40 per 500 mg resulted in an ICER between US $44 and US $28 per life-year saved. Conclusions The addition of 5FC to FLU is cost-effective for cryptococcal meningitis treatment in Africa and, if made available widely, could substantially reduce mortality rates among human immunodeficiency virus–infected persons in Africa., The combination of flucytosine plus fluconazole (FLU) is cost-effective, compared with the commonly used regimen of FLU monotherapy, for cryptococcal meningitis treatment in Africa, with an incremental cost-effectiveness ratio of US $65 per life-year saved at the current price.

Author(s): Mitchell, Leah A; Lopez Espinoza, Fernando; Mendoza, Daniel; Kato, Yuki; Inagaki, Akihito; Hiraoka, Kei; Kasahara, Noriyuki; Gruber, Harry E; Jolly, Douglas J; Robbins, Joan M | Abstract: BackgroundToca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD). Tumor-selective expression of CD converts the prodrug, 5-fluorocytosine (5-FC), into the active chemotherapeutic, 5-fluorouracil (5-FU). This therapeutic approach is being tested in a randomized phase II/III trial in recurrent glioblastoma and anaplastic astrocytoma (NCT0241416). The aim of this study was to identify the immune cell subsets contributing to antitumor immune responses following treatment with 5-FC in Toca 511-expressing gliomas in a syngeneic mouse model.MethodsFlow cytometry was utilized to monitor and characterize the immune cell infiltrate in subcutaneous Tu-2449 gliomas in B6C3F1 mice treated with Toca 511 and 5-FC.ResultsTumor-bearing animals treated with Toca 511 and 5-FC display alterations in immune cell populations within the tumor that result in antitumor immune protection. Attenuated immune subsets were exclusive to immunosuppressive cells of myeloid origin. Depletion of immunosuppressive cells temporally preceded a second event which included expansion of T cells which were polarized away from Th2 and Th17 in the CD4+ T cell compartment with concomitant expansion of interferon gamma-expressing CD8+ T cells. Immune alterations correlated with clearance of Tu-2449 subcutaneous tumors and T cell-dependent protection from future tumor challenge.ConclusionsTreatment with Toca 511 and 5-FC has a concentrated effect at the site of the tumor which causes direct tumor cell death and alterations in immune cell infiltrate, resulting in a tumor microenvironment that is more permissive to establishment of a T cell mediated antitumor immune response.

BACKGROUND: Human NSCs are tumor tropic, making them attractive vehicles for delivery of therapeutics. An immortalized, clonal NSC line was retrovirally transduced to express CD, which converts 5-FC to 5-fluorouracil (5-FU). The primary objectives of this study were to assess the feasibility of serially administering CD-NSCs intracranially via a Rickham catheter and determine the recommended doses for phase II testing (RP2D). METHODS: Adult patients with recurrent high grade gliomas underwent tumor resection or biopsy and placement of a Rickham. CD-NSCs were injected during surgery and thereafter infused through the Rickham every 2 weeks. Three days after each dose of CD-NSCs, patients took 5-FC (and leucovorin—dose level 3 patients only) orally every 6 hours for 7 days. The dose of CD-NSCs was escalated from 50 x 10(6) to 150 x 10(6) using a standard 3 + 3 design. 5-FC and leucovorin doses were 37.5 mg/kg and 25 mg, respectively. A treatment cycle was 28 days, with CD-NSCs administered on days 1 and 15, followed by 5-FC (and leucovorin) on days 4–10 and 18–24. Blood samples were drawn to assess for possible anti-NSC antibody and T cell responses. RESULTS: Fifteen evaluable patients received a median of 2 (range 1–5) cycles of study treatment. One dose-limiting toxicity occurred: grade 3 wound infection. Three patients developed anti-NSC antibodies after receiving 3 doses of NSCs. There was no correlation between these results and use of dexamethasone or number of cycles. Analyses of PK and possible anti-NSC T cell responses are ongoing. Three patients had stable disease for 5 months. CONCLUSIONS: Use of a Rickham to serially administer CD-NSCs intracranially is safe and feasible. Study treatment was well tolerated. There were no clinical signs of immunogenicity to these allogeneic CD-NSCs. The RP2D is 150 million CD-NSCs, 37.5 mg/kg of 5-FC, and 25 mg of leucovorin per dose.

BACKGROUND: The olfactory ensheathing cells (OECs) migrate from the peripheral nervous system to the central nervous system (CNS), a critical process for the development of the olfactory system and axonal extension after injury in neural regeneration. Because of their ability to migrate to the injury site and anti-inflammatory properties, OECs were tested against different neurological pathologies, but were never studied in the context of cancer. Here, we evaluated OEC tropism to gliomas and their potential as a “Trojan horse” to deliver therapeutic transgenes through the nasal pathway, their natural route to CNS. METHODS: OECs were purified from the mouse olfactory bulb and engineered to express a fusion protein between cytosine deaminase and uracil phosphoribosyltransferase (CU), which convert the prodrug 5-fluorocytosine (5-FC) into cytotoxic metabolite 5-fluorouracil, leading to a bystander killing of tumor cells. These cells were injected into the nasal cavity of mice bearing glioblastoma tumors and OEC-mediated gene therapy was monitored by bioluminescence imaging and confirmed with survival and ex vivo histological analysis. All statistical tests were two-sided. RESULTS: OECs migrated from the nasal pathway to the primary glioma site, tracked infiltrative glioma stemlike cells, and delivered therapeutic transgene, leading to a slower tumor growth and increased mice survival. At day 28, bioluminescence imaging revealed that mice treated with a single injection of OEC-expressing CU and 5-FC had tumor-associated photons (mean [SD]) of 1.08E + 08 [9.7E + 07] vs 4.1E + 08 [2.3E + 08] for control group (P

Fungal mycetoma (eumycetoma) represents one of the most difficult infections to appropriately manage. The current recommended treatment is based on extensive surgical debridement combined with prolonged antifungal therapy with ketoconazole or itraconazole. Despite the different phylogenetic positions of black-grain eumycetoma species, they are all treated with the same antifungal agents. The in vitro antifungal susceptibility of coelomycetous eumycetoma agents in the order of Pleosporales presently is largely unknown. Here we determined the in vitro activity of eight antifungal agents against seven species causing human eumycetoma using the Sensititre YeastOne method. High minimum inhibitory concentrations (MICs) were found with fluconazole, caspofungin, flucytosine, and amphotericin B. Voriconazole and posaconazole were found to be active against all species tested. Of the species included in the investigation, MICs of Medicopsis romeroi differed from the rest of the mycetoma causative agents belonging to the order of the Pleosporales. We found significantly lower MICs for amphotericin B and significantly higher MICs for fluconazole, ketoconazole, and itraconazole against this species. Our results emphasised that identification of black grain mycetoma agent is important as well as performing susceptibility testing before starting of antifungal treatment.

Toca 511, a clinical-stage retroviral replicating vector (RRV) encoding an optimized yeast cytosine deaminase (CD) prodrug activator gene, in combination with Toca FC (extended-release 5-fluorocytosine (5-FC)), is designed to produce 5-FU which kills cancer cells and immune-suppressive myeloid cells in the tumor microenvironment, leading to anti-cancer immune activation and long-term survival. This combination treatment is currently under evaluation in an international Phase 2/3 trial in patients with recurrent high-grade glioma. In the present study, we investigated the feasibility of further applying Toca 511 & Toca FC in medulloblastoma, the most common malignant tumor of the pediatric nervous system. Aggressive chemotherapy regimens increase survival in children with this disease; however, systemic toxicity limits efficacy. Here we have initiated the first preclinical studies to evaluate RRV-mediated prodrug activator gene therapy in medulloblastoma. Human medulloblastoma cell lines (HDMB03, DAOY) or patient-derived primary medulloblastoma cells were transduced with RRVs encoding either Emerald green fluorescent reporter (RRV-EMD) or the CD enzyme (RRV-CD). RRV replication in vitro, examined by flow cytometry for EMD fluorescence, showed spread to >90% of cells in culture by day 10 post-inoculation. In contrast to non-transduced or RRV-EMD-transduced controls, RRV-CD-transduced medulloblastoma cells exhibited significant dose-dependent reduction of viability upon exposure to 5-FC, as measured by MTS assay. In vivo, stereotactic intracerebellar implantation of non-transduced HDMB03 cells into nude mice resulted in cell dose-dependent median survival time and eventual 100% lethality at all cell doses tested. In this orthotopic medulloblastoma model, mice with RRV-CD-transduced intracerebellar HDMB03 tumors demonstrated increased survival upon 5-FC treatment, as compared to mice treated with PBS or implanted with non-transduced cells. These results support further investigation of RRV-CD-mediated prodrug activator gene therapy for this pediatric malignancy.

Background Candida auris is an emerging multi-drug-resistant human pathogen. Experimental data on the pathogenicity of C. auris is scarce, especially regarding its virulence compared with C. albicans. Additionally, studies of drug efficacy against C. auris rely on conventional animal models that are laborious and low throughput; alternative, less cumbersome models are desirable. To that end, we developed a C. auris fly infection model. Methods We injected 2-week-old TollI-RXA/Tollr632 female flies with a needle dipped in Candida solutions (108 yeast cells/mL) in the dorsal side of the thorax. Flies were infected with 10 different C. auris strains (source: CDC/FDA) and a C. albicansclinical strain. For drug protection studies, C. auris isolate AR-BANK#0386 [MICs: fluconazole (FLC) > 64, posaconazole (POSA) 0.125–0.25, isavuconazole (ISA) 0.25–1, voriconazole (VRC) 0.5–2 µg/mL)] was used. We assessed survival differences associated with different inocula (107 to 1010 yeast cells/mL) and yeast strains. Moreover, protection conferred by addition of FLC, VRC, ISA, POSA, or FLC combined with 5-FC (flucytosine) and/or nikkomycin Z (NikZ) to fly food was studied. Three independent runs were performed for each experiment. Results A) All C. auris strains and C. albicans exhibited comparable in vitro growth rates. B) All strains of C. auris were similarly more virulent than C. albicans (P < 0.0001), with all flies dying by day 7 post-infection. C) FLC, VRC, ISA, FLC+5-FC, FLC+NikZ, or FLC+NikZ+5-FC-fed flies infected with C. auris #0386 had comparably poor survival outcomes compared with untreated C. auris #0386-infected flies. Interestingly, survival rates were improved in POSA-fed infected flies compared with FLC-treated or untreated infected flies (P < 0.0001). As POSA is a cell-associated drug, we are conducting C. auris phagocytosis assays with Drosophila hemocytes that are co-incubated or not with POSA. Conclusion Drosophila is a promising, fast, and inexpensive in-vivo model to study pathogenesis and drug activity in C. auris candidiasis. Disclosures N. Beyda, Astellas: Scientific Advisor, Grant recipient. D. P. Kontoyiannis, Merck: Consultant, Research support and Speaker honorarium. Pfizer: Consultant, Research support. Astellas: Consultant, Research support and Speaker honorarium. Gilead: Speaker’s Bureau, Speaker honorarium. F2G Inc.: Speaker’s Bureau, Speaker honorarium. Cidara Inc.: Speaker’s Bureau, Speaker honorarium. Jazz Pharmaceuticals: Speaker’s Bureau, Speaker honorarium.

Background. There are no prospective data regarding the management of pulmonary cryptococcosis in the immunocompetent patient. Clinical guidelines recommend oral fluconazole for patients with mild to moderate symptoms and amphotericin B plus flucytosine followed by fluconazole for severe disease. It is unclear whether patients who have histological evidence of Cryptococcus neoformans but negative cultures will even respond to drug treatment. We evaluated and managed a patient whose presentation and course raised important questions regarding the significance of negative cultures, antifungal choices, duration of therapy, and resolution of clinical, serologic, and radiographic findings. Methods. In addition to our experience, to answer these questions we reviewed available case reports and case series regarding immunocompetent patients with pulmonary cryptococcosis for the last 55 years using the following definitions: Definite - Clinical and/or radiographic findings of pulmonary infection and respiratory tract isolation of C. neoformans without other suspected etiologies; Probable - Clinical and radiographic findings of pulmonary infection, histopathologic evidence of C. neoformans, and negative fungal cultures with or without a positive cryptococcal polysaccharide antigen. Results. Pulmonary cryptococcosis resolves in most patients with or without specific antifungal therapy. Clinical, radiographic, and serologic resolution is slow and may take years. Conclusions. Persistently positive antigen titers are most common in untreated patients and may remain strongly positive despite complete or partial resolution of disease. Respiratory fungal cultures are often negative and may indicate nonviable organisms.

BACKGROUND Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, used in combination with 5-fluorocytosine (5-FC) kills tumor by local production of 5-fluorouracil (5-FU), inducing local and systemic immunotherapeutic response resulting in long-term survival after cessation of 5-FC. Toca 511 and Toca FC (oral extended-release 5-FC) are under investigation in patients with recurrent high-grade glioma. Lomustine is a treatment option for patients with high-grade glioma. METHODS We investigated the effects of lomustine combined with Toca 511 + 5-FC in syngeneic orthotopic glioma models. Safety and survival were evaluated in immune-competent rat F98 and mouse Tu-2449 models comparing Toca 511 + 5-FC to lomustine + 5-FC or the combination of Toca 511 + 5-FC + lomustine. After intracranial implantation of tumor, Toca 511 was delivered transcranially followed by cycles of intraperitoneal 5-FC with or without lomustine at the first or fourth cycle. RESULTS Coadministration of 5-FC with lomustine was well tolerated. In F98, combination Toca 511 + 5-FC and lomustine increased median survival, but "cures" were not achieved. In Tu-2449, combination Toca 511 + 5-FC and lomustine increased median survival and resulted in high numbers of cure. Rejection of tumor rechallenge occurred after treatment with Toca 511 + 5-FC or combined with lomustine, but not with lomustine + 5-FC. Mixed lymphocyte-tumor cell reactions using splenocytes from cured animals showed robust killing of target cells in an effector:target ratio-dependent manner with Toca 511 + 5-FC and Toca 511 + 5-FC + lomustine day 10. CONCLUSION The combination of Toca 511 + 5-FC and lomustine shows promising efficacy with no additive toxicity in murine glioma models. Immunotherapeutic responses resulting in long-term survival were preserved despite lomustine-related myelosuppression.

Background : Recent publications suggest the distribution of Candida species causing candiduria may vary geographically, which has implications for the continued efficacy of antifungal therapy and emerging resistance. Aim : To investigate the incidence of Candiduria at a university hospital in the UK. Further, to assess the distribution of species and the accompanying antifungal susceptibility profile, in order to monitor the clinical utility of current antifungal treatment guidelines for candiduria so that patients receive the best possible outcomes from the most up to date care. Design : Retrospective audit. Methods : From 1st January 2005 to 31st October 2014, we retrospectively reviewed 37 538 positive urine cultures recorded in a computerized laboratory results database. Identification and susceptibility testing was performed using the VITEK® 2 fungal susceptibility card (bioMerieux, Marcy d'Etoile, France). Results : In total, 96 cultures were positive for Candida species, of which 69 (72%) were C.albicans , which translates to a prevalence of 2.6 per 1000 positive urine cultures. Candiduria was more common in younger patients, males and catheterized females. We report 94 and 73% of isolates of C.albicans and other non- C.albicans Candida species were susceptible to fluconazole. All isolates were susceptible to amphotericin B. Conclusions : Our results add weight to the evidence supporting current European and North American guidelines recommending fluconazole or amphotericin B for treatment of candiduria, if antifungal treatment is clinically indicated.

To the editor—We appreciate the thoughtful comments raised by Wolbers and Day [1] in response to our article [2]. We agree with their primary point—that our meta-analysis summarized data across studies, and it is therefore best suited to evaluate study-level and not individual-level predictors of outcomes. We should have specified more clearly that our analysis is best suited for drawing inferences about relationships among patient populations, but not about individual patients. Indeed, this point is true for almost all meta-analysis, and it also applies to most individual randomized trials [3]. It is important to note that we did not intend to imply that combination amphotericin B and flucytosine conferred a benefit in patients with cryptococcal meningitis with altered mental status. Rather, in our meta-analysis, we were unable to identify a statistically significant treatment benefit across all published literature, for combination amphotericin B with flucytosine over amphotericin B alone. Although there was some implication of a benefit from adjunctive flucytosine in a subanalysis limited to studies that included patients with altered levels of consciousness, this estimate was not statistically significant (odds ratio = 0.56; 95% confidence interval [CI], .23–1.43) (Figure ​(Figure1).1). As such, we hypothesized in the discussion that populations with altered consciousness might be more likely to benefit from adjunctive flucytosine therapy. If this hypothesis were correct, it would require patient-level data for confirmation. Figure 1. Forrest plot from network meta-analysis comparing odds of early (2-week) and late (10-week) mortality between combination amphotericin B and flucytosine with amphotericin B alone for human immunodeficiency virus-associated cryptococcal meningitis. Abbreviations: ... In their recent large, randomized trial, Day et al [4] demonstrated superiority of combination amphotericin B and flucytosine over amphotericin B alone at 10 weeks. They now present a subanalysis with patient-level data that suggests that those with a normal Glasgow Coma Score (GCS) appeared to derive as much benefit (if not more) from the addition of flucytosine as those with a GCS < 15. Although their study was not powered to detect differences in these subgroups, their data do lend support to the use of flucytosine in individual patients with normal mental status within their study population. It should be noted that the overall mortality in their study was 36% at 10 weeks (and 30% among those who received amphotericin B and flucytosine combination therapy). In contrast, the only other large randomized trial to compare amphotericin B alone with amphotericin B and adjunctive flucytosine found no difference in mortality between groups at 10 weeks (6.7% versus 6.9%; relative risk = 0.97; 95% CI, .46–2.04) [5]. It is noteworthy that this other study was conducted in the United States, excluded comatose patients from enrollment, and had a lower prevalence of participants with altered mental status than the Day et al [4] study (11% vs 28%). We believe that the contrasting data from these 2 studies are in line with our overall conclusion: that current available evidence suggests that the adjunctive use of flucytosine might be beneficial in populations with advanced disease who are at high overall risk for mortality. As Wolbers and Day [4] point out, this does not necessarily mean that patients with normal mental status will not benefit from adjunctive flucytosine, only that populations with low overall risk of mortality are less likely to benefit. Unfortunately, for the time being, it appears that flucytosine is largely only available and in use in the areas of the world where the current data suggest it has the least benefit, whereas populations with the highest mortality often cannot access it [6, 7]. Indeed, the most important conclusion we draw from our study is that more data is required, across a range of patient populations and disease stages, to elucidate which drugs are needed for which patients, and to ensure that the optimal therapies are available to those who need them.

INTRODUCTION: High grade glioma (HGG) remains resistant to standard therapies. Newer approaches, such as immunotherapy, hold great promise. Viral infection and killing of tumor cells can stimulate antitumor immune responses. A Phase 1 clinical study using an investigational retroviral replicating vector, Toca 511, injected intratumorally, in combination with investigational oral Toca FC (extended-release 5-FC) is being conducted. METHODS: The primary purpose of this study is to identify the highest safe and well tolerated dose of Toca 511 administered transcranially into recurrent HGG tumors. The starting dose was 3.9 x 10^6 Transducing Units (TU). Using a standard 3 + 3 design, doses have been escalated at half log intervals to 1.5 x 10^9 TU. 24 subjects were injected using a brain biopsy needle. Addition of gadolinium to the vector with post-operative iMRI suggested variable delivery of vector to the tumor. Subsequently 12 subjects have been treated using CED under real-time MRI-guidance. Monthly cycles of oral 5-FC are started 4 weeks after injection of the vector. RESULTS: 36 subjects across 5 vector dosing cohorts have been studied. Real-time MRI-guidance demonstrated vector delivery with confirmation of vector RNA and CD protein in tumor samples. The injection procedures and vector and 5-FC dosing have been safe and well tolerated to date. No DLTs have been observed. Tumors have been observed to regress in the area around vector infusion and some subjects have experienced improvement in clinical symptoms. A number of subjects have experienced prolonged survival with 8 of the first 17 subjects with Grade 4 tumors surviving more than 1 year. CONCLUSION: This study confirms the feasibility and safety of transcranial, intratumoral delivery of an RRV. Continued dose escalation is planned and may yield further gains in efficacy.

Background. Cryptococcal meningoencephalitis is a lethal infection with relatively few therapeutic options. The optimal dosage of liposomal amphotericin B (LAmB) alone or in combination with flucytosine is not known. Methods. A murine model of cryptococcal meningoencephalitis was used. The fungal density in the brain was determined using quantitative cultures. Pharmacokinetic–pharmacodynamic relationships were determined for LAmB and flucytosine administered alone. The effect of the combination was described using the Greco model and a mathematical model. The results were bridged to humans. Results. Inoculation resulted in hematogenous dissemination and logarithmic growth within the central nervous system. There was histological evidence of multifocal infection throughout the brain. Both LAmB and flucytosine produced a dose-dependent reduction in fungal burden. The effect of the combination of agents in the brain was additive. Bridging studies suggested that a human dosage of LAmB 3 mg/kg/d resulted in a submaximal antifungal effect. Regimens of LAmB 6 mg/kg/d alone, LAmB 3 mg/kg/d plus fl ucytosine 50 mg/kg/d, and LAmB 3m g/kg/d plus flucytosine 100 mg/kg/d all resulted in near-maximal antifungal activity. Conclusions. Potential regimens for further study in clinical trials include LAmB 6 mg/kg/d alone, LAmB 3 mg/kg/d plus flucytosine 50 mg/kg/d, and LAmB 3 mg/kg/d plus flucytosine 100 mg/kg/d.

A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated Delta supAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the Delta supAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive.

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as ‘Hit and Run’ and ‘vector recycling’ which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.

The amination of 1,3-dimethyl-5-fluaro-4-thiouracil in ethanolic medium at elevated temperatures has been investigated. The N(3)-methyl, fixed imino, analogues of 5-fluorocytidine, 1-β-D-arabinofuranosyl-5-fluorocytosine and 1-methyl-5-fluorocytosine were prepared by diazomethane treatment, and some of their properties are described. In particular the basicity method was applied to determine the imino/amino tautomeric equilibrium constants (K(T))for the 5-halogenocytosine nucleosides relative to cytidine, and the possible relevance of these results to mutagenesis by 5-halogenopyrimidine nucleosides.