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Background: Flucytosine is an antifungal agent used in combination with other medicines for the treatment of fungal infections. It was available as intravenous (IV), oral tablet, and capsule formulations up until October 2021, when the IV product, Ancotil, was discontinued with no alternative brands available. Aim: This study aimed to formulate a suitable formulation with appropriate stability data for medium to long‐term nasogastric (NG) administration use. Method: Flucytosine 500 mg tablets (Ancotil) were crushed and suspended in (1) Ora‐Plus (OP) + Ora‐Sweet (OS) and (2) Ora‐Blend (OB) to produce 100 mg/mL suspensions (n = 3 for each suspending base) that were stored at 2–8°C in amber glass bottles until assayed. Appearance, odour and pH, and the concentrations of flucytosine in the suspensions were determined by high‐performance liquid chromatography on days 1, 8, and 15. A subjective assessment of the ease of suspension for NG administration via a size 10fr nasogastric tube (NGT) was also tested. Ethics approval was not required for this research article as it was a stability study and did not contain human participants or human data. Results: One of the three OB suspension bottles demonstrated significant suspension clumping resulting in all OB suspensions being excluded from further analysis. There was no change in appearance, odour or pH with the OP + OS based flucytosine suspensions and they extruded easily through a size 10fr NGT with minimal force. The three OP + OS bottles of flucytosine suspension were stable (>98% at all timepoints assessed) for 15 days at 2–8°C when stored in amber glass bottles. Conclusions: The OP + OS suspensions showed chemical stability for up to 15 days when stored under refrigerated conditions and protected from light, making this a suitable multidose enteral alternative to IV flucytosine. [ABSTRACT FROM AUTHOR]

Background: Relapse of Candida albicans urinary tract infection (UTI) is frequent despite appropriate treatment, as commonly used antifungals such fluconazole and flucytosine are only fungistatics. To improve treatment of Candida UTI and decrease relapses, understanding the long-term metabolic activity and survival of C. albicans in urine containing antifungals at minimal inhibitory concentration (MIC) is needed. Methods: we monitored the survival, metabolic activity and consumption of glucose and proteins by C. albicans using conventional methods and isothermal microcalorimetry (IMC). We also investigated the influence of dead Candida cells on the growth of their living counterparts. Results: For 33 days, weak activity was observed in samples containing antifungals in which C. albicans growth rate was reduced by 48%, 60% and 88%, and the lag increased to 172 h, 168 h and 6 h for amphotericin, flucytosine and fluconazole, respectively. The metabolic activity peaks corresponded to the plate counts but were delayed compared to the exhaustion of resources. The presence of dead cells promoted growth in artificial urine, increasing growth rate and reducing lag in similar proportions. Conclusions: Even with antifungal treatment, C. albicans relapses are possible. The low metabolic activity of surviving cells leading to regrowth and chlamydospore formation possibly supported by autophagy are likely important factors in relapses. [ABSTRACT FROM AUTHOR]

Background: The study and development of fluorouracil metal complexes are important in the development of new synthetic methods and materials with applications in pharmaceuticals, agrochemicals, and materials science. Mothodology: A new Cd(II) compound, (H-5FC) [(H-5FC) Cd Cl ] (1), (where H-5FC is HFlucytosine), was successfully synthesized and crystallized by slow evaporation at room temperature. The compound was characterized by single-crystal X-ray diffraction technique and UV–Visible spectroscopy. Results: The structure shows that the compound constitutes of an independent protonated (H-5FC)+ cation and two protonated flucytosine molecules that coordinate to the Cd(II) ion via an oxygen atom to form a trinuclear [(H-5FC)2Cd3Cl10]2− anionic moieties. The independent protonated (H-5FC)+ bridges the [(H-5FC)2Cd3Cl10]2− anions via N/C–H···Cl/O hydrogen bonds. Supramolecular structure analysis of (1) with the aid of Hirshfeld calculations showed the importance of the H···Cl, O···H, C···Cl, and F···Cl interactions. Their percentages were calculated to be 42.2, 10.3, 6.6, and 8.7%, respectively. The band gap energy of the compound, deduced from the Tauc plot of the absorption spectrum, indicated a wide energy gap of 3.65 eV. [ABSTRACT FROM AUTHOR]

(1) Background: Despite being considered a non-pathogenic yeast, recently, a growing occurrence of Saccharomyces cerevisiae infections has been noted. There is little knowledge about the drug susceptibility of this species. Therefore, the objective of this research was to expand it and determine the drug susceptibility profile of a local collection of clinical isolates of this species. (2) Methods: This study contained 55 clinical isolates identified as Saccharomyces cerevisiae using the MALDI-TOF method. The susceptibility of Saccharomyces cerevisiae was tested to 10 antifungals (amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, micafungin, anidulafungin, caspofungin, and itraconazole) using MICRONAUT-AT tests and manogepix, a new drug, using the microdilution method according to EUCAST. (3) Results: Overall, most strains were classified as sensitive to amphotericin B and flucytosine (MIC ranges of ≤0.03–1 and ≤0.06–0.125, respectively) and also to echinocandins. However, five isolates expressed high MIC values for all of the tested azoles, indicating cross-resistance. The MIC range for manogepix was 0.001–0.125 mg/L, with an MIC50 of 0.03 mg/L and an MIC90 of 0.06 mg/L. (4) Conclusions: The occurrence of resistance to azoles may be a concerning problem and therefore should be investigated further. However, the new antifungal manogepix appears to be an interesting new therapeutic option for treating such infections. [ABSTRACT FROM AUTHOR]

ImportanceNew treatments are needed to improve the prognosis of patients with recurrent high-grade glioma.ObjectiveTo compare overall survival for patients receiving tumor resection followed by vocimagene amiretrorepvec (Toca 511) with flucytosine (Toca FC) vs standard of care (SOC).Design, setting, and participantsA randomized, open-label phase 2/3 trial (TOCA 5) in 58 centers in the US, Canada, Israel, and South Korea, comparing posttumor resection treatment with Toca 511 followed by Toca FC vs a defined single choice of approved (SOC) therapies was conducted from November 30, 2015, to December 20, 2019. Patients received tumor resection for first or second recurrence of glioblastoma or anaplastic astrocytoma.InterventionsPatients were randomized 1:1 to receive Toca 511/FC (n = 201) or SOC control (n = 202). For the Toca 511/FC group, patients received Toca 511 injected into the resection cavity wall at the time of surgery, followed by cycles of oral Toca FC 6 weeks after surgery. For the SOC control group, patients received investigators' choice of single therapy: lomustine, temozolomide, or bevacizumab.Main outcomes and measuresThe primary outcome was overall survival (OS) in time from randomization date to death due to any cause. Secondary outcomes reported in this study included safety, durable response rate (DRR), duration of DRR, durable clinical benefit rate, OS and DRR by IDH1 variant status, and 12-month OS.ResultsAll 403 randomized patients (median [SD] age: 56 [11.46] years; 62.5% [252] men) were included in the efficacy analysis, and 400 patients were included in the safety analysis (3 patients on the SOC group did not receive resection). Final analysis included 271 deaths (141 deaths in the Toca 511/FC group and 130 deaths in the SOC control group). The median follow-up was 22.8 months. The median OS was 11.10 months for the Toca 511/FC group and 12.22 months for the control group (hazard ratio, 1.06; 95% CI 0.83, 1.35; P = .62). The secondary end points did not demonstrate statistically significant differences. The rates of adverse events were similar in the Toca 511/FC group and the SOC control group.Conclusions and relevanceAmong patients who underwent tumor resection for first or second recurrence of glioblastoma or anaplastic astrocytoma, administration of Toca 511 and Toca FC, compared with SOC, did not improve overall survival or other efficacy end points.Trial registrationClinicalTrials.gov Identifier: NCT02414165.

(1) Background: Despite being considered a non-pathogenic yeast, recently, a growing occurrence of Saccharomyces cerevisiae infections has been noted. There is little knowledge about the drug susceptibility of this species. Therefore, the objective of this research was to expand it and determine the drug susceptibility profile of a local collection of clinical isolates of this species. (2) Methods: This study contained 55 clinical isolates identified as Saccharomyces cerevisiae using the MALDI-TOF method. The susceptibility of Saccharomyces cerevisiae was tested to 10 antifungals (amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, micafungin, anidulafungin, caspofungin, and itraconazole) using MICRONAUT-AT tests and manogepix, a new drug, using the microdilution method according to EUCAST. (3) Results: Overall, most strains were classified as sensitive to amphotericin B and flucytosine (MIC ranges of ≤0.03–1 and ≤0.06–0.125, respectively) and also to echinocandins. However, five isolates expressed high MIC values for all of the tested azoles, indicating cross-resistance. The MIC range for manogepix was 0.001–0.125 mg/L, with an MIC50 of 0.03 mg/L and an MIC90 of 0.06 mg/L. (4) Conclusions: The occurrence of resistance to azoles may be a concerning problem and therefore should be investigated further. However, the new antifungal manogepix appears to be an interesting new therapeutic option for treating such infections.

Purpose of Review: Cryptococcal meningitis remains a significant cause of mortality among people living with HIV. This review summarizes current practices and recent advances in the management of cryptococcal meningitis. Recent Findings: Results from recent clinical trials have improved understanding of optimal induction therapy for cryptococcal meningitis, with the most recent data supporting the use of a single high dose of liposomal amphotericin B followed by two weeks of flucytosine and fluconazole. Studies have also demonstrated significantly reduced mortality with therapeutic lumbar punctures in patients with cryptococcal meningitis. Despite advances in management, long-term mortality remains high and may continue even after completion of antifungal therapy, emphasizing the importance of immune restoration in people living with HIV. Summary: Cryptococcal disease remains prevalent among people living with HIV, especially in resource-limited settings. Advances in treatment strategies, as well as increased accessibility to antifungal drugs, screening tests, and antiretroviral therapy, are critical for reducing morbidity and mortality from cryptococcal meningitis. [ABSTRACT FROM AUTHOR]

Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.

The AmBisome Therapy Induction Optimization (AMBITION-cm) trial, conducted in eastern and southern Africa, showed that a single, high dose (10 mg/kg) of liposomal amphotericin B, given with an oral backbone of fluconazole and flucytosine, was noninferior to the World Health Organization (WHO)–recommended regimen of 7 days of amphotericin B deoxycholate plus flucytosine for treatment of human immunodeficiency virus (HIV)–associated cryptococcal meningitis and has been incorporated into WHO treatment guidelines. We believe that the trial also has important implications for the treatment of HIV-associated cryptococcal meningitis in high-income settings. We advance the arguments, supported by evidence where available, that the AMBITION-cm trial regimen is likely to be as fungicidal as the currently recommended 14-day liposomal amphotericin–based treatments, better tolerated with fewer adverse effects, and confer significant economic and practical benefits and, therefore, should be included as a treatment option in guidance for HIV-associated cryptococcal treatment in high-income settings. [ABSTRACT FROM AUTHOR]

Background: Candida albicans (C. albicans) and Candida glabrata (C. glabrata) are mainly associated with vulvovaginal candidiasis (VVC). Management of VVC caused by C. glabrata is particularly challenging due to its inherent reduced susceptibility to fluconazole. In this prospective laboratory-based cohort study, we investigated the effect of pH on in vitro susceptibility of Candida spp. isolates to fluconazole and flucytosine. Methods: Vaginal isolates of C. glabrata, C. albicans, Candida tropicalis (C. tropicalis) and Candida parapsilosis (C. parapsilosis) were tested for susceptibility to fluconazole and flucytosine by Epsilometer test (ETEST) strips on Roswell Park Memorial Institute (RPMI) 1640 medium at pH 7.0 and pH 4.5. Minimum inhibitory concentrations (MICs) were read after 24 h at 35 °C. Results were interpreted according to the European Committee on Antimicrobial Susceptibility testing (EUCAST) guidelines. Results: Mean fluconazole MICs (µg/mL) at pH 4.5 were significantly higher than those at pH 7.0 for C. glabrata (82.55 ± 100.32 versus 14.96 ± 7.71, respectively, p = 0.001) and C. albicans (1.32 ± 7.98 versus 0.96 ± 1.35, respectively, p = 0.017) isolates. A similar effect was not observed with C. tropicalis and C. parapsilosis isolates. In contrast, mean MICs against flucytosine were reduced at pH 4.5 compared to pH 7.0 for all four Candida spp. isolates, with this reduction being statistically significant for C. glabrata and C. parapsilosis isolates. Conclusions: Our data show that the therapeutic efficacy of fluconazole against C. glabrata and C. albicans is reduced at lower (normal vaginal) pH values while the activity of flucytosine is enhanced. Therefore, flucytosine may serve as an effective alternative for the treatment of VVC and recurrent VVC caused by C. glabrata and other Candida spp.

Abstract Background The most appropriate alternative to induction therapy for HIV-associated cryptococcal meningitis (CM) remains unclear when standard treatment is unavailable, inaccessible, intolerable, or ineffective. Methods A prospective, multi-centre cohort study was conducted to analyze the data of 156 HIV-infected patients with CM who were treated with amphotericin B deoxycholate (AmB-D) + flucytosine (5FC), voriconazole (VCZ) + 5FC, or AmB-D + Fluconazole (Flu) as induction regimens. Clinical efficacy, cumulative mortality, and adverse effects were compared among the three treatment groups. Results Fewer deaths occurred by week 4 and week 10 among patients receiving AmB-D + 5FC than among those receiving AmB-D + Flu [4 (5.1%) vs. 8 (16.0%) deaths by week 4; hazard ratio, 1.8; 95% confidence interval [CI], 1.0 to 3.3; p = 0.039; and 8 (10.3%) vs. 14 (28.0%) deaths by week 10; hazard ratio, 1.8; 95% CI, 1.1 to 2.7; p = 0.008, respectively]. AmB-D plus 5FC was found to result in significantly higher rates of cerebrospinal fluid (CSF) culture sterility (57.6% vs. 34% by week 2; 87.9% vs. 70% by week 10; p

ABSTRACT Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes. IMPORTANCE 5-FC is a commonly used antifungal drug for treatment of cryptococcosis caused by Cryptococcus neoformans and C. gattii species complexes. When various amino acids were used as the sole nitrogen source for growth, we found lineage dependent differences in 5-FC susceptibility. Deletion of the classical cytosine permease (FCY2) and deaminase (FCY1) genes caused increased 5-FC resistance in all tested nitrogen sources in C. neoformans but not in C. gattii. Furthermore, we demonstrate that the two GATA family transcription factor genes GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance by regulating the expression of the paralogs of cytosine permease and deaminase genes. Our study not only identifies the new function of paralogs of the cytosine permease and deaminase and the role of their regulatory transcription factors but also denotes the differences in the mechanism of 5-FC resistance among the two etiologic agents of cryptococcosis under different nitrogen sources.

Cryptococcosis is an opportunistic fungal infection caused by members of the two sibling species complexes: Cryptococcus neoformans and Cryptococcus gattii. Flucytosine (5FC) is one of the most widely used antifungals against Cryptococcus spp., yet very few studies have looked at the molecular mechanisms responsible for 5FC resistance in this pathogen. In this study, we examined 11 C. gattii clinical isolates of the major molecular type VGIII based on differential 5FC susceptibility and asked whether there were genomic changes in the key genes involved in flucytosine metabolism. Susceptibility assays and sequencing analysis revealed an association between a point mutation in the cytosine deaminase gene (FCY1) and 5FC resistance in two of the studied 5FC resistant C. gattii VGIII clinical isolates, B9322 and JS5. This mutation results in the replacement of arginine for histidine at position 29 and occurs within a variable stretch of amino acids. Heterologous expression of FCY1 and spot sensitivity assays, however, demonstrated that this point mutation did not have any effect on FCY1 activities and was not responsible for 5FC resistance. Comparative sequence analysis further showed that no changes in the amino acid sequence and no genomic alterations were observed within 1 kb of the upstream and downstream sequences of either cytosine permeases (FCY2-4) or uracil phosphoribosyltransferase (FUR1) genes in 5FC resistant and 5FC susceptible C. gattii VGIII isolates. The herein obtained results suggest that the observed 5FC resistance in the isolates B9322 and JS5 is due to changes in unknown protein(s) or pathway(s) that regulate flucytosine metabolism.

BackgroundVocimagene amiretrorepvec (Toca 511) is an investigational gamma-retroviral replicating vector encoding cytosine deaminase that, when used in combination with extended-release 5-fluorocytosine (Toca FC), results preclinically in local production of 5-fluorouracil, depletion of immune-suppressive myeloid cells, and subsequent induction of antitumor immunity. Recurrent high-grade glioma (rHGG) patients have a high unmet need for effective therapies that produce durable responses lasting more than 6 months. In this setting, relapse is nearly universal and most responses are transient.MethodsIn this Toca 511 ascending-dose phase I trial (NCT01470794), HGG patients who recurred after standard of care underwent surgical resection and received Toca 511 injected into the resection cavity wall, followed by orally administered cycles of Toca FC.ResultsAmong 56 patients, durable complete responses were observed. A subgroup was identified based on Toca 511 dose and entry requirements for the follow-up phase III study. In this subgroup, which included both isocitrate dehydrogenase 1 (IDH1) mutant and wild-type tumors, the durable response rate is 21.7%. Median duration of follow-up for responders is 35.7+ months. As of August 25, 2017, all responders remain in response and are alive 33.9+ to 52.2+ months after Toca 511 administration, suggesting a positive association of durable response with overall survival.ConclusionsMultiyear durable responses have been observed in rHGG patients treated with Toca 511 + Toca FC in a phase I trial, and the treatment will be further evaluated in a randomized phase III trial. Among IDH1 mutant patients treated at first recurrence, there may be an enrichment of complete responders.

Background: Cryptococcal meningitis (CM) is an opportunistic infectious disease that occurs in immunocompromised hosts, not only in patients living with HIV, but also in patients without HIV. The evidence regarding the treatment for CM in patients without HIV is mainly found in small retrospective studies and is extremely limited. Objectives: In the present study, we compared the efficacy of liposomal amphotericin B (L‐AMB) alone and in combination with flucytosine (5‐FC) for the induction treatment of CM in patients without HIV. Patients/Methods: Data were gathered from the Japanese Diagnosis Procedure Combination database obtained from hospitals throughout Japan. The study included 517 patients without HIV but having CM who fulfilled the inclusion and exclusion criteria. We analysed the average effect of adding 5‐FC to L‐AMB treatment using the survival time within 14 days of the diagnosis after adjustment of the baseline clinical characteristics with associations with both selections of the treatment and the prognosis. Results: A total of 146 and 217 CM patients received L‐AMB and L‐AMB with 5‐FC, respectively, within 7 days of diagnosis. L‐AMB with 5‐FC showed better prognosis than L‐AMB on day 14 (mortality 6% vs. 11%, hazard ratio, 0.5775; 95% confidence interval, 0.2748–1.213; p = 0.1, Wald test). Conclusions: From the results of this real‐world database study, we revealed that the combination therapy of 5‐FC on L‐AMB for induction therapy might have an advantage on the survival time of NHNT patients with CM as well as PLHIV patients with CM. [ABSTRACT FROM AUTHOR]

There is an emerging necessity for improved therapies against Candida-related infections, with significant implications for global healthcare. Current antifungal agents, limited in number, target specific pathways, but resistance remains a concern. Flucytosine (5FC) exhibits antifungal activity, particularly against Candida. However, monotherapy efficacy is limited, necessitating combination treatments. Herein, we report PEGylated squalene-based nanocarriers for 5FC loading, aiming to enhance its monotherapy efficacy against Candida strains. The loading of 5FC within micelles was achieved using the ultrasound-assisted solvent evaporation method. The 5FC-loaded micelles, together with non-loaded micelles, were thoroughly characterized and analyzed. STEM and DLS analysis confirmed the core-shell morphology with nanometric dimensions along with improved colloidal stability. The quantification of drug loading efficiency and drug loading capacity was calculated using the UV-Vis technique. The in vitro drug-release studies in simulated physiological conditions showed sustained release within 48 hours. Moreover, the release kinetics calculated using mathematical models showed a Fickian diffusion drug release mechanism in simulated physiological conditions with a slower diffusion rate. The in vitro antifungal activity was tested on Candida albicans, Candida glabrata, and Candida parapsilosis. The results showed improved antifungal activity for the nanotherapeutic and unchanged in vitro toxicity toward normal cells, suggesting promising advancements in 5FC therapy., (© 2024 The Author(s). ChemMedChem published by Wiley-VCH GmbH.)

Vivien Nanfuka,1,* Mkhoi L Mkhoi,2– 4,* Jane Gakuru,2 Richard Kwizera,2 Joseph Baruch Baluku,5 Felix Bongomin,6,7 David B Meya1,2,6 1Infectious Diseases Unit, Kiruddu National Referral Hospital, Kampala, Uganda; 2Infectious Diseases Institute, College of Health Sciences, Makerere University, Kampala, Uganda; 3Mark Wainberg Fellowship Programme, Infectious Diseases Institute, College of Health Sciences, Makerere University, Kampala, Uganda; 4Department of Microbiology and Parasitology, College of Health Sciences, University of Dodoma, Dodoma, Tanzania; 5Division of Pulmonology, Kiruddu National Referral Hospital, Kampala, Uganda; 6Department of Medicine, School of Medicine, College of Health Sciences, Makerere University, Kampala, Uganda; 7Department of Medical Microbiology, Faculty of Medicine, Gulu University, Gulu, Uganda*These authors contributed equally to this workCorrespondence: Felix BongominDepartment of Medical Microbiology, Faculty of Medicine, Gulu University, Gulu, UgandaTel +256-784-523-395Email drbongomin@gmail.comBackground: Cryptococcal meningitis is a leading cause of mortality in advanced HIV disease. A positive cerebrospinal fluid cryptococcal antigen (CrAg) test defines cryptococcal meningitis. Herein, we present a patient with serum and cerebrospinal fluid CrAg negative cryptococcal meningitis, despite a positive cerebrospinal fluid India ink examination and quantitative culture.Case Details: A 56-year-old HIV-positive Ugandan woman, with an undetectable HIV RNA viral load and CD4+ T-cell count of 766 cells per microlitre presented with signs and symptoms consistent with cryptococcal meningitis. Her serum and cerebrospinal fluid CrAg tests were negative despite having a positive cerebrospinal fluid India ink and quantitative culture. On day 1, she was commenced on intravenous amphotericin B deoxycholate (1mg/kg) for 3 days (considering 10 CFU growth of Cryptococcus spp) in combination with oral flucytosine (100mg/kg) for 7 days and then fluconazole 1200mg once daily for the next 11 days. By day 7, she was symptom free and quantitative cerebrospinal fluid culture was negative for Cryptococcus spp. She was discharged on day 9. At 10 weeks (day +40) and 18 weeks (day +72), she was well and adherent to her antiretroviral therapy and on maintenance phase of cryptococcal meningitis on fluconazole at a dose of 400mg once daily.Conclusion: This report alerts clinicians managing patients with HIV-associated cryptococcal meningitis to four uncommon clinical scenarios; first, the possibility of negative serum and cerebrospinal fluid CrAg lateral flow assay results in the context of low cerebrospinal fluid fungal burden in a symptomatic patient. Second, possible occurrence of cryptococcal meningitis in a patient with high CD4 T-cell lymphocyte counts. Third, an early seroconversion of cryptococcal antigenaemia following effective fluconazole therapy. Fourth, an early symptomatic relapse of cryptococcal meningitis albeit negative serum CrAg.Keywords: cryptococcal antigen test, cryptococcal meningitis, amphotericin B, fluconazole, flucytosine, India ink

BackgroundCryptococcal meningitis (CM) is a leading cause of adult meningitis in countries with a high burden of HIV. It has remained a significant cause of morbidity and mortality in Africa despite the extensive rollout of HIV antiretroviral therapy (ART). This study aimed to systematically synthesize the evidence on the prevalence of CM among people living with HIV (PLWH) and its predictors of mortality among adults who are on induction antifungal therapy in Africa.MethodsPubMed/MEDLINE, Embase, and Google Scholar were searched for randomized clinical trials or observational studies published in Africa from 1995 to April 2021. Pooled prevalence of CM among PLWH was calculated using R-studio Version 1.4.1717 software and the data extracted from eligible studies were pooled as percentage with a 95% confidence interval (CI). Predictors of mortality among adults on induction antifungal therapy were synthesized narratively.ResultsOut of 364 studies identified, 17 eligible articles were included in the analysis. The prevalence of CM among PLWH in Africa was 5.11% (95% CI 2.71–9.43%; participants = 10,813; studies = 9; I2 = 97%). In the subgroup analysis, the prevalence was 12.9% (95% CI 4.883–30.0; participants = 533; studies = 3; I2 = 63%) in the years 1995–2010 and 3.18% (95% CI 1.54–6.45; participants = 10,280; studies = 6; I2 = 98%) in the years 2011–2021, with the prevalence significantly decreased by 51% (p = 0.02). Predictors of mortality were fluconazole monotherapy, focal neurological signs, low Glasgow coma scale, and delayed diagnosis of CM at varied timepoint.ConclusionPrevalence of CM has significantly decreased from 1996–2010 to 2011–2021 among PLWH on induction therapy in Africa. Fluconazole monotherapy, focal neurological symptoms, diastolic blood pressure < 60 mmHg, and concurrent tuberculosis coinfection were significant predictors of mortality at 2- and 10-weeks timepoints. CM remains a major concern among PLWH despite increases in ART coverage. Improved access to effective antifungal therapies is needed in Africa for timely initiation of combination induction therapy and better treatment outcomes of PLWH.Systematic review registration[https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=254113], identifier [CRD42021254113].

Background: The most appropriate alternative to induction therapy for HIV-associated cryptococcal meningitis (CM) remains unclear when standard treatment is unavailable, inaccessible, intolerable, or ineffective.Methods: A prospective, multi-centre cohort study was conducted to analyze the data of 156 HIV-infected patients with CM who were treated with amphotericin B deoxycholate (AmB-D) + flucytosine (5FC), voriconazole (VCZ) + 5FC, or AmB-D + Fluconazole (Flu) as induction regimens. Clinical efficacy, cumulative mortality, and adverse effects were compared among the three treatment groups.Results: Fewer deaths occurred by week 4 and week 10 among patients receiving AmB-D + 5FC than among those receiving AmB-D + Flu [4 (5.1%) vs. 8 (16.0%) deaths by week 4; hazard ratio, 1.8; 95% confidence interval [CI], 1.0 to 3.3; p = 0.039; and 8 (10.3%) vs. 14 (28.0%) deaths by week 10; hazard ratio, 1.8; 95% CI, 1.1 to 2.7; p = 0.008, respectively]. AmB-D plus 5FC was found to result in significantly higher rates of cerebrospinal fluid (CSF) culture sterility (57.6% vs. 34% by week 2; 87.9% vs. 70% by week 10; p < 0.05 for both comparisons). However, the differences in CSF culture sterility and mortality between the VCZ + 5FC group and the AmB-D + 5FC group were not statistically significant. VCZ plus 5FC had a significantly advantageous effect on the incidence of new AIDS-defining illness and length of hospital stay, compared with AmB-D plus 5FC. Laboratory adverse events (grade 3 or 4), such as severe anemia, were less frequent with VCZ + 5FC use than with AmB-D combined with 5FC or Flu use.Conclusion: Our results suggest that AmB-D combined with 5FC remains the more efficacious induction regimen compared to AmB-D plus Flu, and that VCZ + 5FC might be a potential alternative when the standard regimen is not readily available, accessible, tolerated, or effective.Clinical Trials: Registration number, ChiCTR1900021195. Registered 1 February 2019, http://www.chictr.org.cn/showproj.aspx?proj=35362 . [ABSTRACT FROM AUTHOR]

Candida species, belonging to commensal microbial communities in humans, cause opportunistic infections in individuals with impaired immunity. Pathogens encountered in more than 90% cases of invasive candidiasis include C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The most frequently diagnosed invasive infection is candidemia. About 50% of candidemia cases result in deep-seated infection due to hematogenous spread. The sensitivity of blood cultures in autopsy-proven invasive candidiasis ranges from 21% to 71%. Non-cultural methods (beta-D-glucan, T2Candida assays), especially beta-D-glucan in combination with procalcitonin, appear promising in the exclusion of invasive candidiasis with high sensitivity (98%) and negative predictive value (95%). There is currently a clear deficiency in approved sensitive and precise diagnostic techniques. Omics technologies seem promising, though require further development and study. Therapeutic options for invasive candidiasis are generally limited to four classes of systemic antifungals (polyenes, antimetabolite 5-fluorocytosine, azoles, echinocandins) with the two latter being highly effective and well-tolerated and hence the most widely used. Principles and methods of treatment are discussed in this review. The emergence of pan-drug-resistant C. auris strains indicates an insufficient choice of available medications. Further surveillance, alongside the development of diagnostic and therapeutic methods, is essential. [ABSTRACT FROM AUTHOR]

Abstract Background 5-fluorocytosine is a pyrimidine and a fluorinated cytosine analog mainly used as an antifungal agent. It is a precursor of 5-fluorouracil, which possesses anticancer properties. To reduce systemic toxicity of 5-fluorouracil during chemotherapy, 5- fluorocytosine can be used as a targeted anticancer agent. Expression of cytosine deaminase by a viral vector within a tumor allows targeted chemotherapy by converting 5-fluorocytosine into the cytotoxic chemotherapeutic agent 5-fluorouracil. However, little is known about the tolerance of 5-fluorocytosine in dogs after prolonged administration. Results In three healthy Beagle dogs receiving 100 mg/kg of 5-fluorocytosine twice daily for 14 days by oral route, non-compartmental pharmacokinetics revealed a terminal elimination half-life of 164.5 ± 22.5 min at day 1 and of 179.2 ± 11.5 min, after 7 days of administration. Clearance was significantly decreased between day 1 and day 7 with 0.386 ± 0.031 and 0.322 ± 0.027 ml/min/kg, respectively. Maximal plasma concentration values were below 100 µg/ml, which is considered within the therapeutic margin for human patients. 5-fluorouracil plasma concentration was below the limit of detection at all time points. The main adverse events consisted of depigmented, ulcerated, exudative, and crusty cutaneous lesions 10 to 13 days after beginning 5-fluorocytosine administration. The lesions were localized to the nasal planum, the lips, the eyelids, and the scrotum. Histological analyses were consistent with a cutaneous lupoid drug reaction. Complete healing was observed 15 to 21 days after cessation of 5-fluorocytosine. No biochemical or hematological adverse events were noticed. Conclusions Long term administration of 5-fluorocytosine was associated with cutaneous toxicity in healthy dogs. It suggests that pharmacotherapy should be adjusted to reduce the toxicity of 5-fluorocytosine in targeted chemotherapy.

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.

IntroductionGene-directed enzyme prodrug therapy (GDEPT) employing the cytosine deaminase (CD) gene, which encodes an enzyme that converts the nontoxic agent 5-fluorocytosine (5-FC) into the chemotherapeutic drug 5-fluorouracil (5-FU), has shown promise both in experimental animals and in clinical trials. Nevertheless, with the transfection systems available presently the percentage of tumor cells incorporating the desired gene is usually too low for successful therapy. We have examined the ability of photodynamic therapy (PDT) to enhance the efficacy of the metabolites, converted from 5-FC by CD gene transfected rat glioma cells.MethodsHybrid tumor cell spheroids consisting of CD poitive and CD negative F98 glioma cells in varying ratios were used as in vitro tumor models. PDT was performed with the photosensitizer AlPcS2a and λ=670nm laser irradiance, both before and after confrontation with 5-FC.ResultsPDT increased the toxicity of 5-FU either as pure drug or derived from monolayers of CD positive cells chalanged with 5-FC. PDT in combination with 5-FC resulted in a significantly enhanced inhibition of hybrid spheroid growth compared to non light treated controls. This was the case even at tumor to producer cell ratios as high as 40:1.ConclusionThe results of the present study show that GDEPT and PDT interact in a synergistic manner over a range of prodrug concentration and tumor to transfected cell ratios. The degree of synergy was significant regardless if PDT treatment was given before or after 5-FC administration. The highest degree of interaction was observed though, when PDT was delivered prior to prodrug exposure.

Disseminated acanthamoebiasis is a rare, often fatal, infection most commonly affecting immunocompromised patients. We report a case involving sinuses, skin, and bone in a 60-year-old woman 5 months after heart transplantation. She improved with a combination of flucytosine, fluconazole, miltefosine, and decreased immunosuppression. To our knowledge, this is the first case of successfully treated disseminated acanthamoebiasis in a heart transplant recipient and only the second successful use of miltefosine for this infection among solid organ transplant recipients. Acanthamoeba infection should be considered in transplant recipients with evidence of skin, central nervous system, and sinus infections that are unresponsive to antibiotics. Miltefosine may represent an effective component of a multidrug therapeutic regimen for the treatment of this amoebic infection.