Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies.

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.

Two-Dimensional Regulation of CAR-T Cell Therapy with Orthogonal Switches.

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.

Clinical and Laboratory Profile of Persons Living with Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome and Histoplasmosis from a Colombian Hospital.

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.

Evaluation of the Specificity of Two Enzyme Immunoassays for Coccidioidomycosis by Using Sera from a Region of Endemicity and a Region of Nonendemicity.

Coccidioidomycosis (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 to Coccidioides spp.), 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.

Notes from the field: Fatal gastrointestinal mucormycosis in a premature infant associated with a contaminated dietary supplement--Connecticut, 2014.

In October 2014, a hospital in Connecticut notified CDC and the Connecticut Department of Public Health of a fatal case of gastrointestinal mucormycosis in a preterm infant. The infant, born at 29 weeks' gestation and weighing 1,400 grams (about 3 pounds), had developed signs and symptoms initially consistent with necrotizing enterocolitis approximately 1 week after birth. Exploratory laparotomy revealed complete ischemia of the gastrointestinal tract from the esophagus to the rectum; a portion of necrotic cecum was sent for microscopic examination. Following surgery, the infant developed multiple areas of vascular occlusion, including a large clot in the aorta, findings not usually associated with necrotizing enterocolitis. The infant died soon after. Histopathology results from the resected cecum revealed an angioinvasive fungal infection consistent with mucormycosis. Gastrointestinal mucormycosis is an extremely rare fungal infection caused by mold in the order Mucorales. It occurs predominantly in low birth weight infants, patients with diarrhea and malnutrition, and those receiving peritoneal dialysis; mortality is 85%. Local investigation revealed that the infant had received a dietary supplement, ABC Dophilus Powder, for 7 days, beginning on day 1 of life.

Exserohilum infections associated with contaminated steroid injections: a clinicopathologic review of 40 cases.

September 2012 marked the beginning of the largest reported outbreak of infections associated with epidural and intra-articular injections. Contamination of methylprednisolone acetate with the black mold, Exserohilum rostratum, was the primary cause of the outbreak, with >13,000 persons exposed to the potentially contaminated drug, 741 confirmed drug-related infections, and 55 deaths. Fatal meningitis and localized epidural, paraspinal, and peripheral joint infections occurred. Tissues from 40 laboratory-confirmed cases representing these various clinical entities were evaluated by histopathological analysis, special stains, and IHC to characterize the pathological features and investigate the pathogenesis of infection, and to evaluate methods for detection of Exserohilum in formalin-fixed, paraffin-embedded (FFPE) tissues. Fatal cases had necrosuppurative to granulomatous meningitis and vasculitis, with thrombi and abundant angioinvasive fungi, with extensive involvement of the basilar arterial circulation of the brain. IHC was a highly sensitive method for detection of fungus in FFPE tissues, demonstrating both hyphal forms and granular fungal antigens, and PCR identified Exserohilum in FFPE and fresh tissues. Our findings suggest a pathogenesis for meningitis involving fungal penetration into the cerebrospinal fluid at the injection site, with transport through cerebrospinal fluid to the basal cisterns and subsequent invasion of the basilar arteries. Further studies are needed to characterize Exserohilum and investigate the potential effects of underlying host factors and steroid administration on the pathogenesis of infection.

Pseudo-outbreak of Lecanicillium and Acremonium species in orthopedic surgery patients.

Acremonium species cause a variety of human infections, while Lecanicillium species have not been reported as human pathogens. We describe a pseudo-outbreak involving both organisms, highlighting the role and limitations of molecular methods in the characterization of rare fungal isolates. Repeated isolation of these fungi from patient tissue samples raises concerns about exogenous contamination in the hospital environment.

A cluster of mucormycosis infections in hematology patients: challenges in investigation and control of invasive mold infections in high-risk patient populations.

Mucormycosis has been reported to be occurring more frequently in hematopoietic stem cell transplant (HSCT) recipients in recent years. We investigated a hospital cluster of mucormycosis cases among patients with hematologic disorders. Case-patients were identified through hospital microbiology and pathology database searches and compared to randomly selected controls matched on underlying disease and hospital discharge date using conditional logistic regression. Environmental assessments, including collection of samples for fungal cultures, were performed. Of 11 case-patients, 6 (55%) had acute myelogenous leukemia and 3 (27%) were allogeneic HSCT recipients. Five case-patients (45%) died. In univariate analysis, case-patients were more likely than controls to have refractory hematologic disease (odds ratio [OR], 13.75; 95% confidence interval [CI], 1.31-689); neutropenia >14 days (OR, 11.50; 95% CI, 1.27-558) or to have received voriconazole prophylaxis (OR, 11.26; 95% CI, 1.11-infinity). A point source was not identified. Factors such as underlying disease state and antifungal prophylaxis type may identify hematology patients at highest risk for mucormycosis. Our investigation highlighted critical knowledge gaps, including strain typing methods, the role of the hospital environment in mucormycosis outbreaks, and hospital environmental infection control measures most likely to reduce exposure of immunosuppressed persons to mucormycetes.

DNA and the classical way: identification of medically important molds in the 21st century.

The advent of the 21st century has seen significant advances in the methods and practices used for identification of medically important molds in the clinical microbiology laboratory. Historically, molds have been identified by using observations of colonial and microscopic morphology, along with tables, keys and textbook descriptions. This approach still has value for the identification of many fungal organisms, but requires expertise and can be problematic in determining a species identification that is timely and useful in the management of high-risk patients. For the increasing number of isolates that are uncommon, atypical, or unusual, DNA-based identification methods are being increasingly employed in many clinical laboratories. These methods include the commercially available GenProbe assay, methods based on the polymerase chain reaction such as single-step PCR, RAPD-PCR, rep-PCR, nested PCR, PCR-RFLP, PCR-EIA, and more recent microarray-based, Luminex technology-based, and real-time PCR-based methods. Great variation in assay complexity, targets, and detection methods can be found, and many of these methods have not been widely used or rigorously validated. The increasing availability of DNA sequencing chemistry has made comparative DNA sequence analysis an attractive alternative tool for fungal identification. DNA sequencing methodology can be purchased commercially or developed in-house; such methods display varying degrees of usefulness depending on the breadth and reliability of the databases used for comparison. The future success of sequencing-based approaches will depend on the choice of DNA target, the reliability of the result, and the availability of a validated sequence database for query and comparison. Future studies will be required to determine sequence homology breakpoints and to assess the accuracy of molecular-based species identification in various groups of medically important filamentous fungi. At this time, a polyphasic approach to identification that combines morphologic and molecular methods will ensure the greatest success in the management of patients with fungal infections.

Population-based surveillance for cryptococcosis in an antiretroviral-naive South African province with a high HIV seroprevalence.

OBJECTIVES: To measure the burden of disease and describe the epidemiology of cryptococcosis in Gauteng Province, South Africa. DESIGN AND METHODS: The study was an active, prospective, laboratory-based, population-based surveillance. An incident case of cryptococcosis was defined as the first isolation by culture of any Cryptococcus species from any clinical specimen, a positive India ink cryptococcal latex agglutination test or a positive histopathology specimen from a Gauteng resident. Cases were identified prospectively at all laboratories in Gauteng. Case report forms were completed using medical record review and patient interview where possible. RESULTS: Between 1 March 2002 and 29 February 2004, 2753 incident cases were identified. The overall incidence rate was 15.6/100 000. Among HIV-infected persons, the rate was 95/100 000, and among persons living with AIDS 14/1000. Males and children under 15 years accounted for 49 and 0.9% of cases, respectively. The median age was 34 years (range, 1 month-74 years). Almost all cases (97%) presented with meningitis. Antifungal therapy was given to 2460 (89%) cases of which 72% received fluconazole only. In-hospital mortality was 27% (749 cases). Recurrences occurred in 263 (9.5%) incident cases. Factors associated with death included altered mental status, coma or wasting; factors associated with survival included employment in the mining industry, visual changes or headache on presentation. CONCLUSIONS: This study demonstrates the high disease burden due to cryptococcosis in an antiretroviral-naive South African population and emphasizes the need to improve early recognition, diagnosis and treatment of the condition.

Outbreak of cutaneous Rhizopus arrhizus infection associated with karaya ostomy bags.

BACKGROUND: We investigated an outbreak involving 2 patients hospitalized at hospital A with cutaneous Rhizopus arrhizus (oryzae) infections of surgically created stomas. METHODS: A cohort study involving all patients having ileostomy or colostomy surgery during the outbreak period (January-April 2005) was performed. Environmental samples, including samples obtained from nonsterile karaya (a plant-derived adhesive) ostomy bags and from select hospital areas, were collected. A point prevalence survey was conducted at 5 unrelated hospitals to assess stoma care practices and mold contamination of karaya ostomy bags outside of hospital A. Zygomycete isolates were identified by standard methods. RESULTS: Infections occurred 7 and 10 days after operations for the 2 patients; 1 patient died. In a 21-patient cohort, receiving the equivalent of > or =0.5 mg/kg per day of prednisone during the week prior to the index date was associated with infection (infection rate, 33% for patients receiving > or =0.5 mg/kg per day of prednisone vs. 0% for patients receiving <0.5 mg/kg per day of prednisone; P=.07). The time to first ostomy bag change was longer for patients with infection (median duration, 8.5 days; range, 7-10 days) than for the 19 patients without infection (median duration, 1.5 days; range, 1-17 days; P=.08). At unrelated hospitals, the median time to first ostomy bag change was 2 days (range, 1-6 days) for 18 patients after ostomy. R. arrhizus was recovered from 10 of 18 karaya ostomy bags from hospital A and from karaya ostomy bags donated from 3 of 5 other hospitals, but it was not recovered from the hospital A environment. CONCLUSIONS: The initial karaya ostomy bag was likely to be the source of Rhizopus infection, and prolonged exposure before the first ostomy bag change might have precipitated infection in these susceptible individuals. Karaya might contain opportunistic molds that can pose an infectious risk among susceptible persons.

Cryptococcus gattii infection: characteristics and epidemiology of cases identified in a South African province with high HIV seroprevalence, 2002-2004.

We describe 46 Cryptococcus gattii-infected persons identified by population-based surveillance conducted in South Africa. Most patients with C. gattii infection presented with meningitis. The mortality rate during hospitalization was 36%. We found no significant differences between persons with and persons without C. gattii infection with regard to clinical presentation, acquired immunodeficiency syndrome diagnosis, concomitant conditions, or prior opportunistic infections. C. gattii isolates had low MICs to the tested antifungal drugs.

Saline-filled breast implant contamination with Curvularia species among women who underwent cosmetic breast augmentation.

BACKGROUND: During December 2000-July 2001, black sediment was noted in saline-filled silicone breast implants of women who had undergone revision surgery at facility A. Curvularia fungus was isolated from implant saline. METHODS: To identify risk factors for contamination with Curvularia species, we performed case-control, retrospective cohort, and laboratory studies and conducted procedural reviews. A case patient was defined as any woman who underwent revision surgery at facility A between January 2000 and June 2001 and had black sediment in her implants. RESULTS: Five patients met the case definition. Contamination was associated with having had surgery performed in operating room (OR) 2 (4/88 vs. 1/140; P=.07) and a longer duration of surgery (P<.001). A longer duration spent in the OR was an additional risk factor (P=.005). Curvularia fungus was isolated from the sterile supply room, where saline bottles had been stored under a water-damaged ceiling, and from the corridor outside OR 2; it was also found more commonly from facility A personnel than from non-facility A personnel (12/34 vs. 4/60; P<.001). Saline was warmed in a cabinet opposite OR 2, which was maintained at negative pressure differentials, then was poured into bowls open to the OR 2 environment before injection into implants. CONCLUSION: Surgeons should always use closed systems to inflate breast implants. Surgery center infection control measures must include moisture control and balanced ventilation systems.

Fungemia caused by Zygoascus hellenicus in an allogeneic stem cell transplant recipient.

Zygoascus hellenicus (Candida hellenica) was isolated from a blood culture from a patient who had received an allogeneic stem cell transplant. The isolate displayed an antifungal susceptibility pattern of decreased susceptibility to fluconazole and itraconazole, high susceptibility to voriconazole, and low susceptibility to caspofungin. The organism was misidentified by a commercial yeast identification system. This is the first reported case of human infection with this rare ascomycetous yeast.

Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program.

To determine the incidence of Candida bloodstream infections (BSI) and antifungal drug resistance, population-based active laboratory surveillance was conducted from October 1998 through September 2000 in two areas of the United States (Baltimore, Md., and the state of Connecticut; combined population, 4.7 million). A total of 1,143 cases were detected, for an average adjusted annual incidence of 10 per 100,000 population or 1.5 per 10,000 hospital days. In 28% of patients, Candida BSI developed prior to or on the day of admission; only 36% of patients were in an intensive care unit at the time of diagnosis. No fewer than 78% of patients had a central catheter in place at the time of diagnosis, and 50% had undergone surgery within the previous 3 months. Candida albicans comprised 45% of the isolates, followed by C. glabrata (24%), C. parapsilosis (13%), and C. tropicalis (12%). Only 1.2% of C. albicans isolates were resistant to fluconazole (MIC, > or = 64 microg/ml), compared to 7% of C. glabrata isolates and 6% of C. tropicalis isolates. Only 0.9% of C. albicans isolates were resistant to itraconazole (MIC, > or = 1 micro g/ml), compared to 19.5% of C. glabrata isolates and 6% of C. tropicalis isolates. Only 4.3% of C. albicans isolates were resistant to flucytosine (MIC, > or = 32 microg/ml), compared to < 1% of C. parapsilosis and C. tropicalis isolates and no C. glabrata isolates. As determined by E-test, the MICs of amphotericin B were > or = 0.38 microg/ml for 10% of Candida isolates, > or =1 microg/ml for 1.7% of isolates, and > or = 2 microg/ml for 0.4% of isolates. Our findings highlight changes in the epidemiology of Candida BSI in the 1990s and provide a basis upon which to conduct further studies of selected high-risk subpopulations.

Resolution of discrepant results for Candida species identification by using DNA probes.

Candida species bloodstream isolates were collected from institutions participating in an active, population-based surveillance for candidemia. Species identifications were performed locally and then confirmed at the Centers for Disease Control and Prevention (CDC) by phenotype-based methods. Discrepancies in species identification between the referring institution and the CDC were noted for 43 of 935 isolates (4.6%). A DNA probe-based species identification system (PCR-enzyme immunoassay [EIA]) was then used to resolve these discrepancies. The PCR-EIA result was identical to the CDC phenotypic identification method for 98% of the isolates tested. The most frequently misidentified species was Candida glabrata (37% of all discrepant identifications). Such misidentifications could lead to the administration of inappropriate therapy given the propensity of C. glabrata to develop resistance to azole antifungal drugs.

Cryptococcosis in a bottlenose dolphin (Tursiops truncatus) caused by Cryptococcus neoformans var. gattii.

We describe the first case of cryptococcosis caused by Cryptococcus neoformans var. gattii in a male Atlantic bottlenose dolphin (Tursiops truncatus). The dolphin showed clinical signs of tachypnea, transient dyspnea, and mild tachycardia and developed multiple hyperechoic nodules, parenchymal consolidation, and thickening of pleura. A diagnosis of bronchopneumonia with pleuritis was made. Itraconazole therapy was implemented for 120 days, and trough levels in serum were within or above the suggested therapeutic range. Titers of cryptococcal antigen in serum increased eightfold during therapy, and the case had a fatal outcome. Necropsy examination findings included enlarged pulmonary lymph nodes and extensive coalescing granulomatous lesions throughout both lungs. Histologic examination revealed numerous, spherical to ellipsoidal, mucicarmine-positive, 3- to 14-microm, encapsulated, budding cells consistent with C. neoformans. Culture of the lung tissue yielded colonies of C. neoformans. The isolate was urease positive and nitrate negative and exhibited phenoloxidase activity. It was positive on canavanine-glycine-bromothymol blue agar. When tested by the Iatron serodiagnostic reagent kit (Iatron Laboratories, Inc.), it was shown to belong to serotype B.