Background: Natural Killer (NK) cells possess potent innate anti-tumor cytotoxicity that can be augmented and focused by engineering with chimeric antigen receptors (CARs). Because NK cells do not express T cell receptors that can direct alloreactivity, they have potential as an off-the-shelf (OTS) cell therapy for the treatment of cancer. We recently demonstrated that a drug-inducible co-activation molecule (inducible MyD88/CD40; iMC) synergizes with transgenic IL-15 to boost CAR-NK cell proliferation, survival and anti-tumor cytotoxic effects (Blood Adv.4:1950 [2020]). Here, we describe the pre-clinical development of an OTS iMC/IL-15-enhanced CAR-NK cell platform targeting B cell maturation antigen (BCMA) for the treatment of multiple myeloma. Methods: NK cells were isolated from peripheral blood mononuclear cells by CD56+ selection, activated with IL-15 and microparticles conjugated with IL-21 and 4-1BB ligand. Activated NK cells were transduced with retrovirus encoding an optimized iMC and IL-15-expressing BCMA CAR construct (iMC-BCMA.z-IL15) where iMC signaling could be activated by exposure to rimiducid (Rim), a small molecule dimerizing ligand. Anti-tumor cytotoxicity and cytokine production was assessed using co-culture assays with control or modified CAR-NK cells against BCMA-expressing myeloma cells (NCIH929, RPMI8226, MM1S, U266 and NALM-6-BCMA). Additional experiments were performed with BCMA-edited cell lines (CRISPR/Cas9) to evaluate the innate cytotoxic potential of GoCAR-NK cells. In vivo anti-tumor efficacy and NK cell expansion was measured using immunodeficient NSG mice engrafted with 1.5 x 106 NCIH929-GFPffluc, MM1S-GFPffluc or THP1-GFPffluc cells followed by i.v. treatment with up to 1 x 107 BCMA GoCAR-NK cells. Tumor and NK cells were tracked via bioluminescence imaging. Results: Following IL-15 and IL-21/4-1BBL microparticle stimulation, NK cells were efficiently transduced (40-70%) and exhibited rapid ex vivo expansion (200-fold in 13 days). iMC-BCMA.CAR-IL15-modified NK cells exhibited potent cytotoxicity against BCMA+target cells compared with mock-transduced NK cells (MM1S, 58±4% versus 17±2%; Nalm-6-BCMA, 61±2% versus 19±6%) after 24 hours. Long-term (7 day) co-culture assays revealed the effect of iMC/IL-15 enhancement on NK cell potency, proliferation and cytokine production where iMC-BCMA.z-IL15-modified NK cells stimulated with Rim showed a >70% increase in tumor-specific killing compared to cells without iMC activation. Further, rimiducid-induced activation led to NK cell persistence and proliferation, 8.1±4.0-fold expansion compared to the start of the coculture. In comparison, there was an 80% reduction mock transduced NK cells or GoCAR-NK cells in cocultures without rimiducid. Induced-MC signaling also drove production of cytokines such as TNF-α, IFN-g (6.6X stimulation with 1 nM Rim relative to no drug), GMCSF, IP-10, and IL-13. In addition, activation of the iMC co-activation protein in combination with IL-15 secretion prevented NK cell exhaustion and led to retained functional activity of the modified GoCAR-NK cells for over 4-weeks in culture. In contrast, unmodified NK cells or modified GoCAR-NK cells without Rim exposure became functionally deficient. Of interest, a comparison of NK and T cells modified with the iMC/IL-15 BCMA CAR construct indicated that CAR-NK cells display more rapid target killing, which is further augmented by iMC-mediated cell signaling in the presence of Rim. Furthermore, GoCAR-NK cells were capable of lysis of BCMA-null target cells due to their innate anti-tumor activity. In vivo efficacy studies showed that neither iMC activation nor IL-15 secretion alone were sufficient to support CAR-NK cell engraftment in NSG mice but, in combination, they resulted in CAR-NK cell expansion and persistence. iMC/IL-15-enhanced BCMA GoCAR-NK cells proliferation was associated with improved control of tumor outgrowth in mice challenged with BCMA+ myeloma cells. Summary: These results indicate that the synergistic activity of iMC signaling combined with transgenic IL-15 production can enhance BCMA-specific CAR-NK cytotoxicity, cytokine production, long-term proliferation and persistence and may improve overall anti-tumor efficacy of a potential OTS cell therapy for the treatment of myeloma. Disclosures Wang: Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Duong:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Guerrero:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Mahendravada:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sharp:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Brandt:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gagliardi:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Foster:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Bayle:Bellicum Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy targeting solid tumors is challenged by inhibitory signals in the tumor microenvironment that may lead to CAR-T exhaustion. Previously, we described a GoCAR platform that uses a rimiducid (Rim)-inducible cytoplasmic costimulatory switch, inducible MyD88/CD40 (iMC), to drive the proliferation, survival, cytokine release and anti-tumor efficacy of T cells expressing a 1st generation CAR. To further understand how iMC signaling improves CAR-T potency, we directly compared iMC-based costimulation to conventional costimulatory domains (i.e., CD28 and 4-1BB) against hematological and solid tumor models. Methods: PBMCs from four donors were activated with anti-CD3/CD28 and transduced with retrovirus encoding 1st generation (CAR.ζ), 2nd generation (CAR.CD28.ζ CAR.4-1BB.ζ or GoCAR (iMC-2A-CAR.ζ) components targeting HER2, CD19 or GD2. Cocultures were established at a 1:1 ratio with antigen-expressing targets and cultures serially passaged weekly against fresh targets for up to six weeks. For in vivo efficacy evaluations NSG mice were engrafted s.c. with 1 × 106 HER2+ OE19-GFPffLuc esophageal carcinoma cells and challenged on day 4 with 5 × 106 CAR-T cells marked with RLuc each monitored by IVIS imaging. Results: 1st generation CAR-T cells produced low levels of IL-2 and IFNγ after the first antigen stimulation and quickly expressed markers of exhaustion including PD-1. 2nd generation CAR-T cells proliferated in early passage but became phenotypically and functionally exhausted following a second stimulation. Conversely, GoCAR-T cells were capable of expanding and sustaining IL-2 production beyond five passages, but only when stimulated with Rim. Lower levels of PD-1, TIM-3, and LAG-3 expression were observed in Rim-treated GoCAR-T cells when compared to 1st gen, 2nd gen and GoCAR-T cells without iMC activation, and PD1 expression remained repressed for six weeks in Rim-treated GoCAR-T cells. In NSG mice engrafted with HER2+ OE19 tumors, Rim treatment drove robust iMC-HER2.ζ CAR-T cell expansion compared to HER2.ζ, HER2.BB.ζ and HER2.28.ζ CAR-T cells over 5 weeks (p < 0.05). iMC-HER2.ζ CAR-T cells significantly enhanced tumor killing with Rim activation relative to vehicle-control and HER2.ζ-expressing T cells days post T cell infusion (tumor radiance = 1.63 × 107 ± 1.7E7 vs 1.40 × 108 ± 5.27E7 vs 1.70 × 108 ± 8.65E7, respectively, p<0.05). When rechallenged with tumor cells at day 35, persistent GoCAR-T cells continued to control tumor growth while 2nd generation CAR-T cells failed to control tumor growth. Furthermore, mice treated with iMC-HER2.ζ CAR-T cells and Rim produced high levels of proinflammatory cytokines including IFN-γ, GM-CSF, and IP-10 consistent with supporting T cell activation and proliferation. Conclusions: iMC activation via Rim infusion provides on-demand control of CAR-T cell signaling to resist CAR-T cell exhaustion, enhance persistence, produce cytokines, and induce antitumor toxicity against solid tumors. Citation Format: MyLinh T. Duong, Aruna Mahendravada, Mary E. Brandt, Kelly L. Sharp, Aaron E. Foster, J. Henri Bayle. Inducible MyD88/CD40 (iMC) enhances CAR-T cell expansion and persistence by overcoming T cell exhaustion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2194.

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting “low” cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns.

Abstract Background Despite impressive efficacy in liquid tumors, improved CAR-T efficacy and persistence appear necessary to control solid tumors, but this increased potency will likely increase the risk of toxicity. Here, we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to cognate ligands. Cell activation is controlled by rimiducid (Rim), which triggers signaling cascades downstream of MyD88 and CD40 via an engineered chimeric protein termed iMC. A rapamycin (Rap)-controlled apoptotic switch, iRC9, is co-expressed, which induces dimerization of caspase-9 active domains to mitigate possible toxicity from excessive CAR-T function. When combined with a first generation CAR in a single vector, these molecular switches allow for specific and efficient regulation of engineered T cells to eliminate HER2+ and PSCA+ cancer cells. Results Transduction of activated T cells using γ-retroviruses encoding the unified dual-switch (DS) HER2- or PSCA-CAR components (SFG-iRC9.2A-CAR.ζ.2A-iMC) yielded efficient transgene expression despite the large insert size (60.5±8.7% CAR+). Greater transduction efficiency was achieved with single-cell producer clones encoding the PSCA DS CAR vector (79.3±2.9% CAR+). When exposed to target antigen in a coculture assay with tumor cells, robust IL-2 and IFN- γ production by DS CAR-T cells was Rim-dependent. Tumor cell killing and T cell expansion were also enhanced by Rim stimulation. In an OE19 tumor-bearing mouse model, Rim stimulation of HER2 DS CAR-T cells significantly enhanced tumor killing (205.8±58.3 mm2 vehicle (veh)-treated vs 55.9±10.9 mm2 Rim-treated, p<0.05) and T cell expansion (4.6E5 average radiance veh-treated vs 1.9E6 Rim-treated). When targeting PSCA+ HPAC cancer cells, PSCA DS CAR-T cells proliferated in a Rim dose-dependent manner and tumor control was maintained even after cessation of Rim treatment. In a second pancreatic tumor model, SU8686, Rim treatment induced long-term tumor control (>80 days). When necessary, deployment of the off-switch (iRC9) rapidly (½ Vmax ~ 8 hours) and efficiently eliminated DS CAR-T cells in a caspase-3 activation assay with real-time (IncuCyte) monitoring, as well as AnnV/PI detection by flow cytometry (3.2±1.0% AnnV+/PI+ veh-treated vs 80.0±5.2% 10nM Rap-treated, p<0.005). In vivo assessment of the iRC9 switch was performed via eGFPluciferase (eGFPfluc)-labeled PSCA dual-switch CAR-T cells in NSG mice. Temsirolimus (Tem; a Rap prodrug) treatment efficiently eliminated PSCA DS CAR-T cells within 6 hours (6.1E4 average radiance veh-treated vs 2.1E4 0.4mg/kg Tem-treated, p<0.0001). Summary Dual-switch CAR-T, a novel platform comprising a CAR combined with regulated costimulation and apoptotic signaling elements, effectively control solid tumor growth and T cell expansion and elimination. This technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells. Citation Format: MyLinh Duong, Eva Morschl, Aruna Mahendravada, Matthew Collinson-Pautz, Mary Brandt, Ming Zhang, Aaron Foster, J. Henri Bayle, David Spencer. A unified dual-switch CAR vector to target solid cancer with controllable "on" and "off" states [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-141.

Abstract Surveillance and outbreak investigations are important epidemiological tools for assessing the frequency, distribution, and determinants of infections. The primary goal of these activities is to identify measures to reduce the burden of disease. This chapter describes examples of surveillance and approaches to outbreak investigations that have formed the basis of fungal infection control measures. However, considerable knowledge gaps exist, new threats are emerging (including antifungal resistance), and healthcare advances are making more people susceptible to severe fungal infections. Expanded surveillance efforts, timely outbreak detection, and effective outbreak investigations are needed to further reduce the burden of fungal infections. This will require confronting challenges that have held back fungal disease epidemiology, including limited clinical suspicion of fungal infections by clinical providers, difficulties in diagnosing fungal infections due to suboptimal diagnostic methods, limited availability of antifungal susceptibility testing and molecular subtyping, and a lack of mandated fungal disease surveillance in most countries.

Abstract Background: While chimeric antigen receptor (CAR)-T immunotherapies have shown remarkable efficacy against leukemias and lymphomas, improved CAR-T efficacy and persistence are needed to overcome solid tumors, without compromising safety. Here, we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to their cognate ligands. Cell activation is controlled by the homodimerizer rimiducid that triggers signaling cascades downstream of MyD88 and CD40 via an engineered chimeric protein termed iMC. A rapamycin-controlled pro-apoptotic switch, iRC9, is co-expressed, which induces dimerization of the caspase-9 domain to mitigate possible toxicity from excessive CAR-T function. When combined with a first generation CAR, these molecular switches allow for specific and efficient regulation of engineered T cells. Methods & Results: T cells were activated and co-transduced with the HER2 GoCAR (SFG-iMC.2A-CAR.ζ) and RapaCIDe (SFG-iRC9.2A-ΔCD19) vectors to generate “Dual-switch GoCAR-T” cells. Combined transduction of RapaCIDe and HER2 GoCAR vectors into T cells did not adversely affect the antitumor efficacy of the GoCAR-T cells, which eliminated OE19 esophageal tumor cells in a 7-day coculture assay at a 1:20 effector to target ratio (3.9±4.3% OE19-eGFPFluc cells remained in GoCAR-modified cultures vs. 1.1±0.1% for the dual-switch GoCAR), and promoted T cell expansion (53.4±9.4% CAR+ for GoCAR vs. 44.6±13.2% for the dual-switch). When challenged in an OE19 tumor-bearing mouse model, rimiducid stimulation of the dual-switch GoCAR-T cells enhanced tumor killing and T cell expansion. Deployment of the off-switch induced fast (½ Vmax ~ 8 hours) and efficient elimination of T cells (Dual-switch GoCAR-T = 94.1% AnnV+/PI+ vs. GoCAR-T = 5.1%) in a caspase-3 activation assay with real-time (IncuCyte) monitoring as well as AnnV/PI detection by flow cytometry. In vivo assessment of the RapaCIDe switch was performed via eGFPluciferase (eGFPfluc)-labeled RapaCIDe-T cells in NSG mice. Rapamycin, but not rimiducid, treatment efficiently eliminated RapaCIDe-T cells within 24 hours, which is similar to the rate observed by the clinically validated rimiducid-regulated CaspaCIDe switch. Summary: Dual-switch GoCAR-T, a novel platform comprising a first-generation CAR combined with regulated costimulation and apoptotic signaling elements, effectively controlled tumor growth and T cell expansion and elimination in vitro and in vivo. This dual-switch technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells. Citation Format: MyLinh T. Duong, Matthew R. Collinson-Pautz, Eva Morschl, Mary E. Brandt, Ming Zhang, Kevin W. Slawin, Aaron E. Foster, J. Henri Bayle, David M. Spencer. Dual-switchGoCAR-T cells: small molecule-regulated “GO” and “STOP” switches to target solid cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-184. doi:10.1158/1538-7445.AM2017-LB-184

Background Improvement of the efficacy and safety of chimeric antigen receptor (CAR)-T immunotherapies requires controlled activation and termination of the T cells when transfused into patients. Here we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to their cognate ligands. T cell costimulation is controlled by the homodimerizer rimiducid that triggers signaling cascades downstream of MyD88 and CD40 via an engineered protein termed iMC. A rapamycin-controlled pro-apoptotic switch (iRC9) that induces dimerization of caspase-9 mitigates possible CAR-T cell toxicity. iRC9 is a chimeric protein comprisingan FKBP-rapamycin binding (FRB) domain in tandem with FKBP12 and caspase-9. This design permits rapamycin, a heterodimerizing ligand, to function as a homodimerizer. When combined with a first generation CD123-specific CAR, these molecular switches allow for controlled, robust expansion of engineered T cells to control acute myelogenous leukemia (AML) in vitro and in vivo combined with a rapid and efficient safety mechanism to block excessive cytokine release. Methods & Results T cells were activated and co-transduced with pSFG-iMC.2A.CD123CAR.ζ and pSFG-iRC9.2A.ΔCD19 vectors to generate Dual-switch (DS) CAR-T cells. Combined transduction of iRC9 and iMC-CAR vectors produced CD123-directed CAR-T cells that eliminated CD123+ THP1 and MOLM13 AML cells, but not CD123- HPAC tumor cells, in a co-culture assay. Cytokine secretion and target cell killing were dependent on the dose of rimiducid (EC50 Deployment of the off-switch induced fast (½ Vmax ~ 8 hours) and efficient T cell elimination of in a caspase-3 activation assay with real-time monitoring by Incucyte microscopy as well as Annexin V detection by flow cytometry (DS CAR-T = 77.6% versus untransduced = 2.2% Annexin V+ when treated with 1 nM rapamycin). In vivo assessment of the suicide switch was performed with eGFP Fluc -labeled CD123 DS CAR-T cells in NSG mice. Rapamycin, but not rimiducid, treatment efficiently eliminated DS CAR-T cells within 24 hours in NSG mice, which is similar to the clinically validated rimiducid-regulated iC9 switch. Importantly, the off-switch was insensitive to high rimiducid concentration, demonstrating that the on-switch regulator does not crosstalk with the safety switch. Summary Dual switch CAR-T, a novel platform comprising a first-generation CAR combined with regulated activation and apoptotic signaling elements, effectively controlled tumor growth and T cell expansion and elimination in vitro and in vivo . This dual switch technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells. Disclosures Bayle: Bellicum Pharmaceuticals: Employment, Equity Ownership. Duong: Bellicum Pharmaceuticals: Employment, Equity Ownership. Lu: Bellicum Pharmaceuticals: Employment. Morschl: Bellicum Pharmaceuticals: Employment, Equity Ownership. Collinson-Pautz: Bellicum Pharmaceuticals: Employment, Equity Ownership. Sharp: Bellicum Pharmaceuticals: Employment, Equity Ownership. Szymanski: Bellicum Pharmaceuticals: Employment, Equity Ownership. Brandt: Bellicum Pharmaceuticals: Employment, Equity Ownership. Slawin: Bellicum Pharmaceuticals: Consultancy, Equity Ownership. Toler: Bellicum Pharmaceuticals: Employment, Equity Ownership. Yvon: Bellicum Pharmaceuticals: Equity Ownership. Foster: Bellicum Pharmaceuticals: Employment, Other: stockholders . Spencer: Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders .

Histoplasmosis is common among persons living with human immunodeficiency virus/acquired immune deficiency syndrome (PLWHA) in Latin America, but its diagnosis is difficult and often nonspecific. We conducted prospective screening for histoplasmosis among PLWHA with signs or symptoms suggesting progressive disseminated histoplasmosis (PDH) and hospitalized in Hospital La María in Medellín, Colombia. The study's aim was to obtain a clinical and laboratory profile of PLWHA with PDH. During 3 years (May 2008 to August 2011), we identified 89 PLWHA hospitalized with symptoms suggestive of PDH, of whom 45 (51%) had histoplasmosis. We observed tuberculosis (TB) coinfection in a large proportion of patients with PDH (35%), so all analyses were performed adjusting for this coinfection and, alternatively, excluding histoplasmosis patients with TB. Results showed that the patients with PDH were more likely to have Karnofsky score ≤ 30 (prevalence ratio [PR] = 1.98, 95% confidence interval [CI] = 0.97-4.06), liver compromised with hepatomegaly and/or splenomegaly (PR = 1.77, CI = 1.03-3.06) and elevation in serum of alanine aminotransferase and aspartate aminotransferase to values > 40 mU/mL (PR = 2.06, CI = 1.09-3.88 and PR = 1.53, CI = 0.99-2.35, respectively). Using multiple correspondence analyses, we identified in patients with PDH a profile characterized by the presence of constitutional symptoms, namely weight loss and Karnofsky classification ≤ 30, gastrointestinal manifestations with alteration of liver enzymes and hepatosplenomegaly and/or splenomegaly, skin lesions, and hematological alterations. Study of the profiles is no substitute for laboratory diagnostics, but identifying clinical and laboratory indicators of PLWHA with PDH should allow development of strategies for reducing the time to diagnosis and thus mortality caused by Histoplasma capsulatum.

ABSTRACTCoccidioidomycosis (CM), a serious life-threatening fungal infection endemic to arid regions of the western United States and Mexico, can be challenging to diagnose in a timely manner. Commercially developed enzyme immunoassays (EIAs) (from Meridian Biosciences and Immuno-Mycologics [IMMY]) have provided faster, simpler means for serodiagnosis; however, independent evaluations have questioned EIA specificity, particularly IgM-positive/IgG-negative results. This study was conducted to evaluate EIA specificity among persons residing in Puerto Rico (n= 534), where CM is not endemic (who were not likely to have been exposed toCoccidioidesspp.), compared to blood bank donors residing in Arizona (n= 1,218), where CM is endemic. Upon comparing serum reactivity between Puerto Rico and Arizona, the Meridian EIA showed a significant difference in IgG reactivity (0.37% versus 3.6%;P< 0.001) but not IgM reactivity (3.4% versus 2.4%;P= 0.31). No IgM-/IgG-reactive sera were detected among sera from Puerto Rico, compared to 7 (0.57%) sera from Arizona. Similar results were observed using the IMMY EIA, although significantly (P= 0.03) fewer IgM-reactive sera from Arizona were observed, compared to the Meridian EIA. EIA-reactive sera were also evaluated by immunodiffusion before and after 3- to 4-fold concentration of the sera. These results demonstrate that elevated IgG EIA reactivity is present in sera from healthy individuals in regions of endemicity and that IgM EIA reactivity observed in sera from individuals residing outside regions of endemicity is most likely nonspecific. Other criteria, including clinical and microbiological evaluations, should be taken into account when interpreting results from surveillance studies and other reporting measures.

Transplant recipients are at a high risk for developing invasive fungal infections. The agents of phaeohyphomycosis are environmental molds found worldwide, and they cause a broad spectrum of disease including skin and subcutaneous lesions, pneumonia, central nervous system disease, fungemia, and disseminated disease. Using data from the Transplant Associated Infection Surveillance Network (TRANSNET), we evaluated patients with proven and probable phaeohyphomycosis. Centers collected data on demographics, co-morbid conditions, clinical features, treatment, and three-month mortality. Fifty-six patients with phaeohyphomycosis were identified from 15 centers, comprising 26 stem cell transplant (SCT) and 30 solid organ transplant (SOT) recipients. Median time to diagnosis post-transplant was 358 days (SCT 100 days; SOT 685 days; P = <.001). The most frequent pathogen was Alternaria species (32%). Disseminated disease was found in 55.4%. Cutaneous infection was more common in SOT (53.3% vs 23.1%; P = .021), while pulmonary disease was more common in SCT (57.7 vs. 26.7; P = .019). Voriconazole (44.6%) and amphotericin B preparations (37.5%) were the most common antifungal therapies. Overall mortality was 25% and was higher in SCT than in SOT (42% vs 10%; P = <.001). A wide variety of organisms encompass phaeohyphomycosis contributing to varying types of infection in transplant recipients. Site of infection, time to disease, and mortality varies significantly between SCT and SOT recipients. Lipid formulations of amphotericin B and voriconazole were the most common antifungals used to treat this disorder.

ABSTRACT Early availability of antifungal susceptibilities can ensure timely institution of targeted therapy in candidemia, which can improve patient outcomes. This study prospectively determines the agreement between the results of direct testing of antifungal susceptibilities from blood culture bottles by disk diffusion and Etest and the results of standardized susceptibility testing methods; direct testing would allow susceptibility results to be available 1 to 2 days earlier. A total of 104 blood cultures with different Candida species (28% C. albicans, 27% C. parapsilosis, 26% C. tropicalis, etc.) were evaluated between January 2012 and May 2013 for agreement of fluconazole, voriconazole, and amphotericin B susceptibility results by disk diffusion. Agreement in MICs obtained by Etest was determined for fluconazole (21 isolates), voriconazole (28 isolates), amphotericin (29 isolates), and caspofungin (29 isolates). The kappa scores for categorical agreement were highest for fluconazole by disk diffusion (0.902, standard error [SE] = 0.076) and Etest (1.00, SE = 0.218) and for amphotericin B by disk diffusion (1.00, SE = 0.098). The Pearson correlation (r) of zone diameters was strongest for fluconazole (0.69) and amphotericin (0.70) and moderate for voriconazole (0.60), and the Pearson correlation of MICs was strongest for fluconazole (0.94) and caspofungin (0.88). However, the moderate correlation of amphotericin MICs with zone diameters (−0.42) precludes the use of amphotericin B disk diffusion for susceptibility testing. There were no very major errors; however, there were 1 (1%) major and 5 (4.8%) minor errors with disk diffusion and 4 (13.3%) minor errors with Etest. Thus, antifungal disk diffusion directly from blood culture bottles is a rapid and easy method for fluconazole and voriconazole susceptibility testing for timely tailoring of candidemia therapy.

Exserohilum rostratum was the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range and E. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection of E. rostratum in body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive for E. rostratum to 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-D-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data.