New formulation and delivery method of Cryphonectria parasitica for biological control of chestnut blight.

AIMS: This study aimed to develop a new formulation of Cryphonectria parasitica hypovirulent mycelium suitable for inoculations of tall trees from the ground. Cryphonectria parasitica hypovirulent strains are widely used for biological control of chestnut blight. However, it is often inconsistent and ineffective not only for biological reasons but also because the current manual application of hypovirulent strains on adult plants is difficult, time-consuming and expensive. Here, we propose an improved formulation and more effective mode of application of hypovirulent strains, which could boost chestnut blight biocontrol. METHODS AND RESULTS: The Cp 4.2H hypovirulent strain was formulated as mycelium discs with polyethylene glycol and hydroxypropyl methylcellulose, loaded into lead-free pellets that are used as carriers to inoculate cankers on chestnut stems by shooting. The formulation of mycelium did not hamper its viability which was stable, with an estimated shelf life of 72 days at 6 ± 1°C. The inoculum effectiveness was confirmed ex planta and in planta in a small-scale pilot study in field, where formulated mycelium discs of hypovirulent strain Cp 4.2H were inoculated by airgun shot method into the chestnut bark. In planta, Cp 4.2H was recovered in 37% of bark samples taken around the inoculated points 1 year after the treatment. CONCLUSIONS: We demonstrated that the proposed airgun shooting inoculation method of C. parasitica hypovirulent strain formulated as mycelium discs is suitable for treatment of adult chestnut trees. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method could be a valid alternative to the traditional manual technique of chestnut biocontrol. The main advantages are the cost-effectiveness and the ease to treat high-positioned, otherwise unreachable cankers both in orchards and forests.

Prospective study of fungaemia in a single cancer institution over a 10-y period: aetiology, risk factors, consumption of antifungals and outcome in 140 patients.

Over a 10-y period (1989-99) we prospectively evaluated all patients with fungaemia among 16,555 admissions (21,004 blood cultures) at a national cancer referral institution in the Slovak Republic. A prospective protocol was completed on 140 patients with fungaemia, which was then analysed in terms of aetiology, clinical characteristics, potential risk factors and outcome. The most frequently isolated organism was C. albicans, in 75 patients (52.9%), followed by non-albicans Candida spp. in 45 patients (32.1%). Non-Candida spp. yeasts represented 16 episodes in 16 patients (11.4%). Moulds caused 4 episodes in 4 patients (3.6% of all fungaemias) and all were caused by Fusarium spp. Mucositis (p = 0.025), > or = 3 positive blood cultures (p = 0.02), acute leukaemia (p = 0.00001), neutropenia (p = 0.0015), quinolone prophylaxis (p < 0.000005) and breakthrough fungaemia (p = 0.004) during prophylaxis with fluconazole (p = 0.03) and itraconazole (p = 0.005) were significantly more associated with non-Candida than C. albicans spp. Furthermore, attributable mortality was higher in the subgroup of non-Candida than C. albicans spp. (50.0 vs. 18.7%, p < 0.02). The only independent risk factor for inferior outcome was antifungal therapy of < 10 d duration (odds ratio 2.1, 95% confidence interval, p < 0.001). Aetiology, neutropenia and mucositis were not independent risk factors for higher mortality in multivariate analysis; however, they were risk factors for inferior outcome in univariate analysis (p < 0.05-0.005).

Antibiotic use and development of resistance in blood culture isolates: 8 years of experience from a cancer referral center.

The consumption of antimicrobial agents in a Slovakian national cancer institute from 1989-1996 was compared with resistance rates in clinically significant blood culture isolates. We observed an increase in resistance of viridans streptococci to penicillin and of enterococci to ampicillin. Resistance to vancomycin and teicoplanin was stable over the entire period despite a 20-fold increase in vancomycin consumption. Nor did we observe increased resistance to ofloxacin despite a 10-fold increase in consumption. Consumption of aminoglycosides and resistance levels were both stable. A different situation was observed with third-generation cephalosporins, where resistance of Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter spp. to ceftazidime and cefotaxime increased with increasing consumption. Resistance of Enterobacteriaceae to cefotaxime and ceftazidime was stable. Resistance to imipenem did not change significantly. However, the number of Stenotrophomonas maltophilia bacteremias increased significantly after imipenem was introduced in 1991. Because of improved outcome in bacteremia, an increased incidence of both gram-negative and gram-positive bacteremia led to only a slight increase in associated mortality.

Nosocomial candidaemias due to species other than Candida albicans in cancer patients. Aetiology, risk factors, and outcome of 45 episodes within 10 years in a single cancer institution.

Forty-five cases of fungaemia due non-albicans Candida spp. (NAC) in a single National Cancer Institution within 10 years were analysed for aetiology, risk factors and outcome. There had been 12 cases of fungaemia that were due to C. krusei, 14 due to C. parapsilosis, 7 due to C. (T.) glabrata, 6 to C. tropicalis, 2 to C. guillermondii, 2 to C. lusitaniae, 1 to C. stellatoidea, and 1 to C. rugosa. Comparison of 45 NAC fungaemia with 75 episodes of C. albicans fungaemia revealed differences only in two risk factors: previous empiric therapy with amphotericin B (16.0 vs 2.2%, P<0.01) appeared more frequently in cases of C. albicans fungaemia, and prior prophylaxis with fluconazole (8.9 vs 0%, P<0.02) was conversely more frequently observed with NAC. The incidence of other risk factors, such as underlying disease, chemotherapy, antibiotic prophylaxis or therapy, treatment with corticosteroids, catheter insertion, mucositis, cytotoxic chemotherapy, and neutropenia, was similar in both groups. There was no difference either in attributable or in overall mortality between NAC and C. albicans fungaemia in our cancer patients.

Nosocomial Candida krusei fungemia in cancer patients: report of 10 cases and review.

The risk factors, therapy and outcome of ten cases of fungemia due to Candida krusei, appearing during the last 10 years in a single national cancer institution, are analyzed. Univariate analyses did not find any specific risk factors in comparison to 51 Candida albicans fungemias appearing at the same institution and with a similar antibiotic policy. Association with prior fluconazole prophylaxis was not confirmed because only one case appeared in a patient previously treated with fluconazole. However, attributable and crude mortality due to C. krusei fungemias was higher than for C. albicans fungemia. The authors review 172 C. krusei fungemias published within the last 10 years to compare with the incidence, therapy and outcome of C. krusei fungemia from our cancer institute.

Hematogenous trichosporonosis in cancer patients: report of 12 cases including 5 during prophylaxis with itraconazol.

Twelve cases of Trichosporon spp. fungemias occurring in a national cancer institution within 10 years are described. The trend of hematogenous trichosporonosis within the last 10 years is increasing. While no cases occurred in 1988-1991, after 1991, Trichosporon spp. was the most common species among non-Candida spp. fungemias in 1993-1997. The 12 cases of fungemia included 5 that started while the patients were receiving prophylaxis with oral itraconazole, and 2 appeared despite empiric therapy with amphotericin B. Five of the 12 fungemias were catheter associated. Risk factors for fungemia were: central venous catheter, broad-spectrum antibiotics (third-generation cephalosporins plus aminoglycoside); all but 1 had neutropenia and were receiving antineoplastic chemotherapy. All but 2 of the patients died of systemic fungal infection (83.3% mortality). Amphotericin B was administered to all but 1 patient, who was not treated because he died the day after his culture was found to be positive for T. beigelii, before antifungals were administered. All cases infected with T. pullulans were catheter related, and all these patients died. One of the remaining 9 fungemias was caused by T. capitatum (Blastoschizomyces capitatus), and 8 by T. beigelii. Only 2 patients were cured, 1 with a combination therapy with amphotericin B plus fluconazole, and 1 with amphotericin B monotherapy. Several risk factors (neutropenia, acute leukemia, prior therapy or prophylaxis with antifungals and catheter as source of fungemia, breakthrough fungemia) were significantly associated with Trichosporon spp. fungemia, in comparison to 63 C. albicans candidemia occurring in the same period at the same institution. Attributable mortality of hematogenous trichosporonosis was also significantly higher (83.3% vs. 15.8%, P<0.001) than that of hematogenous candidiasis.

Invasive infections due to Clavispora lusitaniae.

Three cases of Clavispora lusitaniae invasive fungal infections are reported. All three infections appeared in cancer patients presented with fungaemia, one additionally with meningitis. Two of them were breakthrough -- they developed during therapy with conventional amphotericin B with a dose of 0.5 mg kg(-1) day(-1) . All three were cured: two with intravenous fluconazol and one with an increasing dose (1 mg kg(-1) day(-1)) of amphotericin B. In one of two breakthrough cases the sensitivity of the strain to antifungals was tested against antifungal agents and showed in vitro resistance to amphotericin B (MIC 2 eta g ml(-1)).

Documented fungal infections after prophylaxis or therapy with wide spectrum antibiotics: relationship between certain fungal pathogens and particular antimicrobials?

Antibiotics are known to be one of the major risk factors for fungal infection. We investigated whether there was a relationship between particular documented fungal infections and therapeutically or prophylactically administered antimicrobials in 105 patients with fungemia or histologically proven invasive aspergillosis or fusariosis. Out of 105 patients, 82.9% received antimicrobials affecting anaerobic microbial gut flora such as: imipenem, vancomycin, ceftazidime, metronidazole, clindamycin or ampicillin-sulbactam. In addition, 44.5% of patients had received prophylaxis with ofloxacin. 31.5% of Candida albicans fungemias occurred despite empiric therapy with amphotericin B and 21.1% during prophylaxis with azoles. The incidence of C. albicans infections (fungemias) was significantly higher (58.9% vs 33.7%, p<0.04) in patients receiving antibiotics not affecting anaerobic gut flora such as ofloxacin, an aminoglycoside or azithromycin. On the other hand, patients treated with third generation cephalosporins, carbapenems, glycopeptides, and broad spectrum penicillins were more likely to develop proven invasive Aspergillus spp. infection (27.9% vs 5.3%, p<0.001) in comparison to those treated with antimicrobials which preserve anaerobic gut flora.

Bacteremia due to multiresistant gram-negative bacilli in neutropenic cancer patients: a case controlled study.

The aim of this study was to see if multiresistant Gram-negative bacteremias (MRGNB) are associated with specific risk factors and/or higher mortality in comparison to sensitive GNB (SGNB). Both groups, 51 patients and 102 controls, were matched for sex, age, underlying disease and neutropenia. In addition there were no significant differences in the incidence of cytotoxic chemotherapy administered, vascular catheter insertion and catheter as source of bacteremia and etiology of bacteremia. The proportion of Klebsiella-Enterobacter, Pseudomonas aeruginosa, Acinetobacter spp. and Stenotrophomonas maltophilia was similar in both groups. Prior surgery (21.6% vs 7.6%, p<0.02) was significantly associated with SGNB. Previous prophylaxis with quinolones (45.1% vs 24.5%, p<0.045), and prior therapy with broad spectrum antibiotics (41.2% vs 27.5%, p<0.05) were significantly more frequently observed among patients than controls. Patients with bacteremia due to MRGNB were also significantly more frequently infected with resistant bacteria. Attributable mortality was similar (15.7% vs 13.75%, NS) in both groups, however cure rates were lower among MRGNB patients. Crude mortality was higher among patients (35.3% vs 13.75%, p<0.01) in comparison to controls. In conclusion, prior antimicrobial prophylaxis and therapy with several classes of antimicrobials represents a significant risk for development of resistance. Mortality due to multiresistant Gram-negative bacteremias was higher in comparison to bacteremias due to susceptible organisms.

Bacteremia and fungemia in pediatric versus adult cancer patients after chemotherapy: comparison of etiology, risk factors and outcome.

One hundred and eighteen (118) episodes of bacteremia and fungemia in children with cancer were compared to 401 episodes of bacteremia and fungemia in adults with cancer to assess differences in etiology, risk factors and outcome. A retrospective univariate analysis was performed of all episodes of bacteremia in national pediatric and adult cancer institutions appearing in 1990-1996. A total of 519 episodes of bacteremia were assessed and compared. Both cancer centers differed in prophylactic antibiotic policies. About 50% of adults but less than 5% of children received quinolone prophylaxis during neutropenia, even though the empiric antibiotic therapeutic strategy was similar. There were differences in etiology between the groups: staphylococci and Stenotrophomonas maltophilia were more frequently observed in children (P<0.01), Pseudomonas aeruginosa and Acinetobacter spp. in adults (P<0.05). Gram-positive bacteremia was surprisingly more commonly observed in adults (65.7% vs 33.3%, P<0.01). Mixed polymicrobial bacteremia occurred more commonly in adults (31.8% vs 7.6%, P<0.001) than in children. Analysis of risk factors did not observe differences in risk factors except for underlying disease (acute leukemia was more frequently observed in children -48.3% vs adults 33.7%, P<0.05 and prophylaxis: (prior prophylaxis with quinolones was more common in adults (47.5%) than in children (2.5%) P<0.0001). Overall and attributable mortality in pediatric bacteremia was significantly lower than in adults (P<0.03).

Predictors of mortality in bacteremic cancer patients: retrospective analysis of 64 deaths occurring among 262 bacteremic episodes.

A total of 262 bacteremic episodes were observed in cancer patients in a single cancer institution during the last 7 years, and the recorded outcome was death in 65. The 65 patients who died (24.8% overall mortality) were divided retrospectively into two subgroups: (a) those who died of underlying disease with bacteremia (45 cases, 16.9% crude mortality) and (b) those who died of bacteremia (20 patients, 7.7% attributable mortality). Comparison of several risk factors in subgroups of patients who achieved a cure (197 cases) and of those who died and whose deaths were attributable (20 cases) revealed six risk factors that were associated with attributable mortality: (1) chemotherapy-induced neutropenia (P < 0.03), (2) Acinetobacter/Stenotrophomonas spp. bacteremias (P < 0.001), (3) liver failure (P < 0.001), (4) inappropriate therapy (P < 0.0001), (5) organ complications (P < 0.003) and (6) multiresistant organisms (P < 0.001). Enterococci and Pseudomonas aeruginosa, surprisingly, were found more frequently in those who died of an underlying disease with bacteremia than among patients who were cured (17.6% vs 7.6%, P < 0.05 and 29.1% vs 13.8%, P < 0.02). Those who died of infection had higher numbers of positive blood cultures, with 2.05 per episode, than did those who died of underlying disease with bacteremia (1.82) or those who were cured (1.51). Other risk factors, such as underlying disease, type of chemotherapy, origin of bacteremia, age, and catheters did not predict either overall or attributable mortality within the study group.

Bacteremia due to methicillin-resistant staphylococci occurs more frequently in neutropenic patients who received antimicrobial prophylaxis and is associated with higher mortality in comparison to methicillin-sensitive bacteriemia.

Bacteriemia due to coagulase-negative staphylococci (CNS) resistant to methicillin and sensitive only to glycopeptides in 220 cancer patients was prospectively analyzed for risk factors and outcome. A group of 33 cases of bacteriemia with CNS-sensitive only to glycopeptides was compared with a group of 187 cases with CNS sensitive to methicillin. All cases appeared in two affiliated major cancer institutes in Bratislava with the same antibiotic policy. Univariate analysis showed differences in recorded risk factors: acute leukemia (48 vs. 33%, P < 0.05), neutropenia (57 vs. 32%, P < 0.045), previous prophylaxis with quinolones (30 vs. 11%, P < 0.01) and penicillin-V (15 vs. 3%, P < 0.02) and previous colonisation with CNS (27 vs. 3%, P < 0.01) were more frequently associated with bacteriemia resistant to methicillin and sensitive only to glycopeptides. Attributable mortality was also higher in this subgroup in comparison to bacteriemias with CNS sensitive to methicillin (12 vs. 3%, P < 0.05) however, overall mortality was similar. Bacteriemias due to CNS caused by sensitivity only to glycopeptides occurred more frequently in neutropenic patients (1), with acute leukemia (2), receiving quinolone and penicillin prophylaxis (3), and previously colonized (4), patients and had worse prognosis in comparison to those with methicillin-sensitive staphylococcal bacteriemias.

Nosocomial breakthrough fungaemia during antifungal prophylaxis or empirical antifungal therapy in 41 cancer patients receiving antineoplastic chemotherapy: analysis of aetiology risk factors and outcome.

Forty-one episodes of breakthrough fungaemia occurring over a 7.5 year period in the National and St Elizabeth's Cancer Institutes in Bratislava, Slovakia, were analysed. Five of them occurred during prophylaxis with fluconazole (one Torulopsis glabrata, one Hansenula anomala, two Candida krusei and one Candida parapsilosis), ten with itraconazole (three Trichosporon pullulans, one Trichosporon beigelii, one Cryptococcus laurentii, three Candida albicans and two T. glabrata), 11 during prophylaxis with ketoconazole (one Candida norvegenesis, one C. parapsilosis, one C. krusei, one Candida tropicalis, five C. albicans, one Candida stellatoidea and one C. laurentii and 15 during empirical therapy with amphotericin B (ten C. albicans, two T. beigelii and three Candida lusitaniae). The most frequent risk factors for breakthrough fungaemia were neutropenia, previous therapy with multiple antibiotics and recent catheter insertion. Comparing these episodes with 38 non-breakthrough fungaemias (appearing at the same institute in the same period) differences in certain risk factors were noted: breakthrough fungaemias were more frequently observed in patients with acute leukaemia (39.0% vs 5.2%, P < 0.001), mucositis (34.2% vs 13.1%, P < 0.05), prophylaxis with quinolones (58.5% vs 15.8%, P < 0.0001) and catheter-associated infections (29.3% vs 2.6%, P < 0.003). In this subgroup overall mortality (36.6% vs 28.8%) or early attributable mortality (22.0% vs 23.6%) were not significantly different.

Comparison of the E test with broth microdilution for antifungal susceptibility testing of yeasts.

The susceptibilities of 26 recent invasive clinical isolates to amphotericin B (AMP), 5-flucytosine (5FC), fluconazole (FLU) and itraconazole (ITR) were determined by a broth microdilution modification of the NCCLS M27P method and also by E test. Using breakpoint criteria each result was classified as either sensitive (S), intermediate (I) or resistant (R). Taking the optical density (OD)80 as the standard, the results were further classified into major (M) or minor (m) errors. E test: AMP = 0M 0m, 5FC = 0M 5m, FLU = 1M 12m, ITR = 1M 5m errors. Minimal inhibitory concentrations (MIC): AMP = 0M 2m, 5FC = 0M 0m, FLU = 3M 4m, ITR = 1M 7m errors. The E test was quick and relatively simple to perform. Results using the E test compared favourably with those of the OD80 and it should be suitable for the routine susceptibility testing of yeasts to antifungal agents.

Eight-year surveillance of non-albicans Candida spp. in an oncology department prior to and after fluconazole had been introduced into antifungal prophylaxis.

From 1989 until 1996, during the last 8 years, the proportion of Candida (C.) krusei, and other non-albicans Candida spp. isolated from surveillance cultures and from sterile body sites, was analyzed among 13,758 admissions in a National Cancer Institute. During these admissions a total of 9,042 isolates were prospectively collected from surveillance cultures, and 126 from blood cultures. The proportion of C. krusei among all organisms was 12.7% to 16.5% in 1989 through 1991, i.e., before fluconazole was introduced into prophylactic protocols. After the introduction of fluconazole into prophylaxis in acute leukemia in 1992 the incidence of C. krusei was 7.9% to 8.6% during 1994 to 1996. After 5 years of using this drug for prophylaxis, the incidence of C. krusei was lower than before this drug was introduced in our institute. Among yeasts, the most frequently isolated pathogen was still Candida albicans (72.2% of all isolated fungal organisms). Among molds, Aspergillus spp. was the most frequently isolated agent. Analyzing the etiology of proven fungal infections (fungemias) confirmed by positive blood cultures, C. albicans was the most common causative organism in 53.8% of cases. The incidence of fungemia due to Torulopsis (C.) glabrata and C. krusei before and after fluconazole introduction did not change. Of 126 organisms isolated from blood cultures, there was no increase in T. (C.) glabrata or C. krusei after introduction of fluconazole for prophylaxis and therapy, and the quoted 6.4% of fungemic episodes remained stable with an incidence of 1 fungemia/year since 1991. The proportion of C. krusei and C. glabrata among Candida spp. was decreasing in our center between 1989 and 1996. Also, the proportion of non-albicans Candida spp. among isolates decreased from 25.7% in 1990 to 11.9% in 1996.

Fungaemia due to Fusarium spp. in cancer patients.

Five cases of fungaemia due to Fusarium spp. in cancer patients are described. Two were breakthrough cases, despite ongoing therapy with amphotericin B. Three were caused by Fusarium solani, one by F. oxysporum and one by F. dimerum. Four patients died, three of them despite therapy with amphotericin B for between 5-37 days. We describe only the second reported case of F. dimerum fungaemia. Since 1972, 93 cases of systemic infection with Fusarium spp. have been described: 43 had positive blood cultures and the overall mortality was 72%.