Prospective study of Candida species in patients at a comprehensive cancer center.

Since most nosocomial systemic yeast infections arise from the endogenous flora of the patient, we prospectively evaluated the species stratification and antifungal susceptibility profile of Candida spp. associated with heavy colonization and systemic infection in patients at Memorial Sloan-Kettering Cancer Center in New York. A total of 349 Candida isolates were obtained from 223 patients during the later half of 1998. Cancer was the most common underlying disease, occurring in 91% of the patients, including 61.8% with organ and 23.7% with hematological malignancies; 4.4% of the patients had AIDS. Candida albicans was the predominant species (67.3%); among 114 non-albicans Candida spp., C. glabrata (45.6%) was the most frequent, followed by C. tropicalis (18.4%), C. parapsilosis (16.6%), and C. krusei (9.6%). The overall resistance to triazole-based agents among all yeast isolates was 9.4 and 10.8% for fluconazole and itraconazole, respectively. A total of 5% of C. albicans strains were resistant to triazole antifungals, whereas 30.8 and 46.2% of C. glabrata strains were resistant to fluconazole (MIC > or = 64 microg/ml) and itraconazole (MIC > or = 1 microg/ml), respectively. A significant association was observed between prior treatment with triazole and isolation of fluconazole-resistant C. albicans (P = 0.005, OR 36), although this relationship was not seen in C. glabrata isolates (P = 0.4). This study reinforces the importance of periodic, prospective surveillance of clinical fungal isolates to determine appropriate prophylactic, empiric, and preemptive antifungal therapy for the highly susceptible patient population.

Antimicrobial activities of squalamine mimics.

We investigated the antimicrobial properties of compounds with structural features that were designed to mimic those of squalamine, an antibiotic isolated from the stomach of the dogfish shark. The mimics, like squalamine, are sterol-polyamine conjugates. Unlike squalamine, the mimics were simple to prepare, at high yield, from readily available starting materials. Several squalamine mimics showed activity against gram-negative rods, gram-positive cocci including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and fungi. Some had little or no hemolytic activity. The hydrophobicity of the sterol backbone and the length and the cationic charge of the side chains appeared to be critical determinants of activity. One of the squalamine mimics, SM-7, was bactericidal against Escherichia coli, Pseudomonas aeruginosa, and S. aureus; its activity was decreased by divalent or monovalent cations and by bovine serum albumin. Subinhibitory concentrations of SM-7 markedly enhanced the antimicrobial activity of rifampin against gram-negative rods. These results suggest that the compounds may disrupt an outer membrane of gram-negative rods. Squalamine mimics are a new class of broad-spectrum antimicrobial agents. The antagonism of their activity by serum and albumin and their hemolytic properties may limit their use as systemic agents. The squalamine mimics, because of their potencies, broad spectra of antimicrobial activity, and potential for systemic toxicity, appear to be good candidates for development as topical antimicrobial agents.

Activity of voriconazole (UK-109,496) against clinical isolates of Aspergillus species and its effectiveness in an experimental model of invasive pulmonary aspergillosis.

Voriconazole, a new azole antifungal agent, showed potent activity against clinical isolates of Aspergillus spp. in vitro. For A. fumigatus, the MIC range was < 0.03 to 0.5 microgram/ml and the MIC at which 90% of isolates are inhibited was 0.25 microgram/ml. In an experimental model of invasive pulmonary aspergillosis which mimics infection in humans, oral voriconazole at dosages of 30 mg/kg of body weight per day significantly delayed or prevented mortality.

Efficacy of prophylactic aerosol amphotericin B lipid complex in a rat model of pulmonary aspergillosis.

Invasive pulmonary aspergillosis remains an important cause of morbidity and mortality among transplant recipients and patients receiving cancer chemotherapy. The lipid-associated formulation of amphotericin B (AmB), AmB lipid complex (ABLC), was evaluated for its prophylactic efficacy when it was administered as an aerosol in a rat model of pulmonary aspergillosis. Aerosol ABLC (aero-ABLC), in doses from 0.4 to 1.6 mg/kg of body weight given 2 days before infection, significantly delayed mortality compared to the mortality of rats given placebo (P < 0.001). At day 10 postinfection, 50% of rats in the 0.4-mg/kg group and 75% of rats in the 1.6-mg/kg group were alive, while all control animals had died. In a second trial aero-ABLC was more effective than an equivalent dose of aerosol AmB (aero-AmB) in prolonging survival, with 100% survival at day 14 postinfection in the ABLC group, compared to 62.5% survival in the AmB group. Mean concentrations of AmB in lungs were 3.7 times higher at day 1 (P < 0.002) and almost six times higher at day 7 (P < 0.001) after treatment with aero-ABLC than after treatment with a similar dose of aero-AmB. We conclude that aero-ABLC provided higher and more prolonged levels of the parent compound in the lungs than aero-AmB and was more effective in delaying mortality from aspergillosis in this model.

Induction of antibodies reactive with GM2 ganglioside after immunization with lipopolysaccharides from Camplyobacter jejuni.

Ganglioside GM2, expressed on the surface of some human cancers, is a promising target for immune therapy, since GM2 antibodies are cytotoxic, can be induced in humans by vaccination, and the presence of GM2 antibodies is associated with a better prognosis in melanoma patients. In our efforts to induce long-lived, cytotoxic GM2 antibodies, we investigated lipopolysaccharides (LPS) containing "GM2-like" oligosaccharides. LPS were prepared from Campylobacter jejuni serotypes O:1, O:23, or O:36 (all sharing the oligosaccharide structure GalNAcbeta1-4Gal(113NeuAc)-Hex with ganglioside GM2), and tested for their ability to induce GM2-reactive antibodies. Immunization of NZW rabbits (2 animals per vaccine) with LPS from C. jejuni serotype O:1 in Freund's adjuvant resulted in production of high-titer IgG antibodies reactive with purified bovine brain GM2 in ELISA, dot-blot immune strains and immune thin-layer chromatography, and with GM2 derived from various human tumors by immune thin-layer chromatography. These rabbit antibodies bound to cancer cell lines expressing GM2 on their cell surface, as determined by mixed hemadsorption assays, mediating strong antibody-dependent cellular cytotoxicity (ADCC) with tumor cells expressing cell-surface GM2. Antibodies induced by vaccination with C. jejuni serotype O:1 were higher-titer (IgG ELISA titer > 1:60,000) than antibodies induced by immunization with purified GM2 (IgG ELISA titer > 1:200). Immunization with LPS from C. jejuni serotype O:36 resulted in production of moderately high-titer IgM and low-titer IgG GM2 antibodies. Immunization with LPS from C. jejuni serotype O:23 did not elicit GM2-reactive antibodies. No clinical symptoms were observed in animals immunized with these LPS preparations, with purified GM2 ganglioside, or with LPS derived from C. jejuni serotype O:19 (containing a GM1-like oligosaccharide). Our results indicate that lipopolysaccharides sharing carbohydrate epitopes with gangliosides may be useful immunogens for inducing antibodies to ganglioside antigens.

Aerosol and parenteral pneumocandins are effective in a rat model of pulmonary aspergillosis.

The pneumocandins are semisynthetic analogs of echinocandin-like compounds that have shown efficacy in animal models of systemic candidiasis, disseminated aspergillosis, and pneumocystis pneumonia. However, the most common form of Aspergillus infection in susceptible patients is pulmonary aspergillosis, which was not directly tested in the mouse models used in the past. We have evaluated three pneumocandins, L-693,989, L-731,373, and L-733,560, in a rat model of pulmonary aspergillosis. Male Sprague-Dawley rats were treated for 2 weeks with cortisone and tetracycline and fed a low-protein diet before being inoculated via the trachea with 10(6) conidia of Aspergillus fumigatus H11-20. In the absence of drug treatment, the animals developed a progressive, rapidly fatal bronchopneumonia. All three pneumocandins at doses of 5 mg/kg (intraperitoneally [i.p.] every 12 h [q12h]) were effective in delaying mortality in this model. Survival at day 7 postinfection was 20% among controls (n = 10 for all groups), while it was 60, 80, and 90% in groups that were treated with L-693,989, L-731,373, and L-733,560, respectively. In another trial, survival at day 7 postinfection was 25% among controls (n = 8 for all groups); it was 87.5% in a group treated with amphotericin B (0.5 mg/kg i.p. q12h) and was 100% in a group treated with L-733,560 (0.625 mg/kg i.p. q12h). In a separate trial, aerosol L-693,989 administered 2 h before infection (5 mg/kg) delayed mortality. Eight of the 10 animals treated with aerosol L-693,989 survived for 7 days, whereas only 2 of 10 control animals survived. We conclude that the pneumocandins we tested were highly effective in an animal model of pulmonary aspergillosis.

Restriction fragment length polymorphism analysis of clinical isolates of Mycobacterium haemophilum.

Mycobacterium haemophilum is an emerging opportunistic pathogen, and since 1989, infections caused by this organism have been identified more frequently in the New York City area than in any other region of the United States. A DNA fingerprinting method, based on restriction fragment length polymorphisms (RFLPs) was developed. A genomic library of M. haemophilum isolate 1A was constructed; screening the library yielded a recombinant strain that incorporated a genetic element present in multiple copies in the M. haemophilum genome. This clone was used to produce a probe for RFLP analyses of PvuII digests of genomic DNA. We used this probe to determine the RFLP patterns of 43 clinical isolates of M. haemophilum from 28 patients. A total of six distinct patterns were observed. Two patterns, designated types 1 and 2, accounted for 91% of the infections in patients from the New York City area. Two isolates from Arizona had identical patterns but were distinct from those of New York isolates, and an isolate from Israel, the type strain, had another distinct pattern (type 6). The type 6 pattern was also seen in a recent isolate from Norway. All of the type 1 isolates and 60% of the type 2 isolates were recovered from patients with AIDS in the New York City area. This molecular subtyping method should provide a useful tool for epidemiological studies and may help identify the associated risk factors, vehicles, and possible reservoirs of this newly emerging pathogen.

Activities of antimicrobial agents against clinical isolates of Mycobacterium haemophilum.

Mycobacterium haemophilum, first described in 1978, can cause severe infections of skin, respiratory tract, bone, and other organs of immunocompromised patients. There is no standardized antimicrobial susceptibility test, and for the 27 reported cases, a variety of test methods have been used. This paper reports the in vitro test results for 17 isolates of M. haemophilum recovered from 12 patients in the New York City area. MICs of 16 antimicrobial agents were determined in microtiter trays containing Middlebrook 7H9 broth plus 60 microM hemin, inoculated with 10(6) CFU of the organism per ml and incubated at 30 degrees C for 10 days. Ethambutol, ethionamide, tetracycline, cefoxitin, and trimethoprim-sulfamethoxazole were inactive against initial isolates from the 12 patients. Isoniazid was weakly active with a MIC for 50% of strains tested (MIC50) of 8 micrograms/ml and a MIC90 of > 32 micrograms/ml. Three quinolones, ciprofloxacin, ofloxacin, and sparfloxacin, were moderately active with MIC50s of 2 to 4 micrograms/ml and MIC90s of 4 to 8 micrograms/ml. Amikacin and clofazamine were active with MIC90s of 4 and 2 micrograms/ml, respectively. Clarithromycin was the most active macrolide with a MIC90 of < or = 0.25 microgram/ml. The MIC90 of azithromycin was 8 micrograms/ml, and the MIC90 of erythromycin was 4 micrograms/ml. The rifamycins were active with a MIC90 of 1 microgram/ml for rifampin and one of < or = 0.03 micrograms/ml for rifabutin. For a second isolate from the skin of one patient and a isolate from an autopsy culture of the spleen of a second patient, MICs of rifampin and rifabutin were > 16 microgram/ml, whereas initial isolates were inactivated by low concentrations of the rifamycins. Both patients had been treated for several months with several antimicrobial agents, including a rifamycin.

Azithromycin, rifabutin, and rifapentine for treatment and prophylaxis of Mycobacterium avium complex in rats treated with cyclosporine.

Azithromycin, rifabutin, and rifapentine were used to treat or prevent disseminated Mycobacterium avium complex (MAC) infections produced in rats immunosuppressed with cyclosporine. Animals with bacteremic infections were treated 1 week after intravenous inoculation with 10(7) CFU of MAC with azithromycin, 100 mg/kg of body weight administered subcutaneously for 5 days and then 75 mg/kg on Monday, Wednesday, and Friday, or with rifabutin or rifapentine, 20 mg/kg administered intraperitoneally on Monday through Friday. All three drugs showed efficacy after 1 and 2 months. Rifabutin cleared the organisms from tissues more rapidly than azithromycin or rifapentine. To approximate prophylaxis, treatment was started 2 weeks before intravenous inoculation with 10(4) organisms. MAC infections were undetectable in treated animals after 4 months, while control animals had disseminated infections. These findings support the rationale for clinical trials of treatment and prophylaxis with these agents. The cyclosporine-treated rat appears to be a useful model in which to evaluate compounds for the treatment and prophylaxis of disseminated MAC infections.

Pneumocystosis.

In much of the world, pneumocystosis remains the most common life-threatening opportunistic infection among patients with HIV disease. The infection is caused by Pneumocystis carinii--an organism whose identity as a fungus or parasite is still debated. What is no longer debated, after a decade of AIDS, is that pneumocystosis is almost entirely preventable and eminently treatable. Understanding has improved concerning when prophylaxis should be initiated. It is also recognized that, at least with the agents available today, antiretroviral therapy alone will not prevent pneumocystosis. Sputum induction and the use of monoclonal antibodies have modestly improved our ability to diagnose the infection; however, invasive procedures are still required for most patients, and unusual presentations of the disease, such as cavitary lesions, apical infiltrates, pneumothoraces, and extrapulmonary infection, are not infrequently seen. For treatment, trimethoprim-sulfamethoxazole and intravenous pentamidine remain the mainstays; oral therapy with dapsone and trimethoprim can be as effective as conventional therapy in mild disease, permitting treatment on an outpatient basis. Adjunctive steroids are useful for treatment of moderate to severe pneumocystosis, but clinicians should be alert to the possibility of activation of other latent infections during and after courses of steroids. Both aerosol pentamidine and trimethoprim-sulfamethoxazole are effective prophylaxis. The latter appears to be more effective and costs much less, but the results of comparative trials are not yet available. More data are also needed on the safety, efficacy, and relative advantages of dapsone for prophylaxis. The first decade of the AIDS epidemic has been a decade of progress against pneumocystosis. In the next decade, the emergence of new technologies for diagnosis and of new agents for prophylaxis and treatment will bring us closer to the goal of controlling this serious infection.

Progressive disseminated infection with Mycobacterium avium complex after intravenous and oral challenge in cyclosporine-treated rats.

Male Sprague-Dawley rats, immunosuppressed with cyclosporine (CsA), developed disseminated infection after intravenous or oral challenge with Mycobacterium avium complex (MAC). Disseminated infection leading to bacillemia could be established after intravenous inoculation with as few as 5 x 10(3) organisms. When CsA was not given or when CsA was stopped 1 month after infection, animals cleared the bacilli from blood and tissue. Animals developed disseminated infection after oral challenge with as few as 10(6) organisms. Persistent bacillemia occurred when organisms in the spleen exceeded 10(7). Differences in virulence among strains were observed. Infected tissues showed histopathologic changes similar to those seen in patients with AIDS. The CsA-treated rat is a new model that appears useful for studies of the virulence of MAC strains and the pathogenesis of disseminated MAC infection.

Model of recurrent pulmonary aspergillosis in rats.

Male Sprague-Dawley rats were treated with cortisone acetate and fed a low-protein diet for 3 weeks. At the end of week 2, animals were infected intratracheally with 10(5) conidia of Aspergillus fumigatus H11-20. Despite discontinuation of steroids and the low-protein diet 1 week after the infection, 94% of controls died of invasive pulmonary aspergillosis within 3 weeks postinfection. When rats were treated with a single dose of 1.6 mg of aerosolized amphotericin B per kg of body weight 48 h prior to the infection, mortality was reduced to 11% within 3 weeks postinfection. Despite apparent good health and rapid weight gain, all survivors showed multiple lesions in histopathological sections of the lungs, and 10(3) to 10(4) CFU of aspergilli was recovered from cultures of their lungs. With discontinuation of immunosuppression, the infection was slowly cleared; however, when cortisone acetate was restarted during week 5, reactivation of progressive invasive pulmonary aspergillosis was observed. On the basis of these results, we conclude that a single low dose of aerosolized amphotericin B prophylaxis is effective in preventing an exogenous aspergillus infection of the lung. Additional therapy is needed to prevent recurrent infection caused by endogenous aspergilli when immunosuppression is resumed.

Combination therapy in a model of pulmonary aspergillosis.

The current treatment for pulmonary aspergillosis, amphotericin B, is toxic and not always effective. This study was done to evaluate combinations of amphotericin B with other agents in an animal model of pulmonary aspergillosis. Sprague-Dawley rats were treated with cortisone acetate, infected intratracheally with 10(6) spores of Aspergillus fumigatus, and followed daily for survival. Mortality among controls started on day 2, and it was 80% by day seven, whereas therapy with amphotericin B resulted in survival of all animals. When given alone, ketoconazole, 5-fluorocytosine and rifampin did not improve survival. The combination of ketoconazole with amphotericin B resulted in complete antagonism. When animals received a combination of aerosol amphotericin B prophylaxis two days prior to infection followed by treatments with SCH39304 or itraconazole seven days after infection, survival rates were superior as compared to animals that had received aerosol prophylaxis only. The combinations of either 5-fluorocytosine or rifampin with amphotericin B were not better than amphotericin B alone. While combinations with 5-fluorocytosine or rifampin appear not to offer any advantage over therapy with amphotericin B alone, additional studies to further evaluate the role of azoles in combination therapy are needed.

Pneumothorax in AIDS.

OBJECTIVE: To determine risk factors for the development of pneumothorax in patients with the acquired immunodeficiency syndrome (AIDS). DESIGN: Prospective cohort study. SETTING: Tertiary care center. PATIENTS: Of 1030 patients with AIDS who were followed at Memorial Sloan-Kettering Cancer Center between 1 January 1980 and 30 September 1989, 20 (2%) developed pneumothorax that was unrelated to trauma or a pulmonary procedure. RESULTS: Of 20 patients with AIDS who presented with pneumothorax, 19 had compelling evidence of concurrent Pneumocystis carinii pneumonia. Using bivariate analysis, patients receiving aerosol pentamidine prophylaxis (relative risk, 17.6) and those with a history of P. carinii pneumonia (relative risk, 14.5) were more likely to develop pneumothorax. By Mantel-Haenszel stratified analysis, aerosol pentamidine use was a statistically significant risk factor independent of a history of P. carinii pneumonia. The pneumothorax-related mortality rate was 10% and there was considerable morbidity. CONCLUSIONS: Patients with AIDS at the highest risk for developing pneumothorax are those with a history of P. carinii pneumonia who are receiving aerosol pentamidine prophylaxis but who nevertheless develop P. carinii pneumonia. The benefits of aerosol pentamidine prophylaxis in these patients far outweigh this risk. Pneumocystis carinii pneumonia should be considered as the most likely diagnosis in any patient with AIDS who develops a pneumothorax.

In vitro activity of the new semi-synthetic polypeptide cilofungin (LY121019) against Aspergillus and Candida species.

The in vitro activity of cilofungin (LY121019), a new semi-synthetic antifungal agent was evaluated. Potent activity was seen against Candida albicans and Candida tropicalis, with almost identical MIC and MFC results, whereas no activity was seen against any isolates of Candida parapsilosis or three Aspergillus spp. However, MICs were dependent on medium and test conditions chosen. It is concluded that cilofungin has good activity against some medically important yeasts in vitro, and that its in vitro activity depends on the method used.

Aerosolized pentamidine: effect on diagnosis and presentation of Pneumocystis carinii pneumonia.

STUDY OBJECTIVE: To determine the effect of previous aerosolized pentamidine therapy on diagnosis and presentation of Pneumocystis carinii pneumonia. DESIGN: A retrospective study. SETTING: A tertiary care hospital. PATIENTS: Fifty-two consecutive patients with P. carinii pneumonia and underlying infection with the human immunodeficiency virus (HIV) who had bronchoscopy. Twenty-one patients who were on aerosolized pentamidine therapy served as the study group. Thirty-one patients who had not received the drug served as the control group. MEASUREMENTS AND MAIN RESULTS: The yield of bronchoalveolar lavage for P. carinii pneumonia was 62% for the study group and 100% for the control group (P less than 0.05). This lower yield was significant for the subset of patients having their first episode of P. carinii pneumonia. The yield of transbronchial biopsy was similar for both groups of patients (81% compared with 84%). The yield of bronchoscopy was not influenced by use of zidovudine. Review of lavage specimen slides suggested that there may be fewer organisms present in patients receiving aerosolized pentamidine. An atypical roentgenographic presentation of upper lobe predominant infiltrates was seen in 38% of the study patients and 7% of the control patients. In addition, pneumothoraces and cystic changes were also frequently seen in the study patients. Gallium scans, when done, were also atypical in the study group. Markers of the severity of disease, however, were similar in both groups. CONCLUSION: The yield of bronchoalveolar lavage for P. carinii pneumonia in HIV-infected patients is lower in patients receiving aerosolized pentamidine. Unusual roentgenographic presentations and atypical gallium scans are also found in this setting.

Amphotericin B blunts erythropoietin response to anemia.

Amphotericin B causes a normochromic, normocytic anemia thought to be mediated by direct marrow toxicity or suppression of erythropoietin production. Serial hemoglobin, hematocrit, amphotericin B, and erythropoietin levels were determined before, during, and after completion of amphotericin B therapy for three patients without significant renal disease or active hematologic malignancy. Patients with systemic fungal diseases treated with itraconazole served as controls. Serum erythropoietin levels were determined by radioimmunoassay and amphotericin B by high-performance liquid chromatography. Despite anemia in all amphotericin B-treated patients, erythropoietin levels declined or remained relatively constant during therapy while erythropoietin levels in controls were appropriate for the degree of anemia. Within 2 weeks of completion of amphotericin B treatment, two patients had increasing erythropoietin levels in response to anemia. Amphotericin B appears to suppress but not abolish the erythropoietin response to anemia; this effect disappears quickly after discontinuation of the drug.

Pharmacokinetics of aerosol amphotericin B in rats.

The distributions of amphotericin B (AmB) in tissue were compared after intraperitoneal or aerosol administration. Rats were sacrificed 24 h after receiving single or repeated daily doses; AmB concentrations in tissues were determined by high-performance liquid chromatography. After intraperitoneal doses of 4 mg/kg of body weight per day for 7 days, mean concentrations of AmB were 122.7, 55.2, and 4.31 micrograms/g in the spleen, liver, and lung, respectively. After aerosol doses (aero-AmB) of 1.6 mg/kg per day, the mean concentrations of AmB in the lung were 2.79 micrograms/g after a single dose and 9.88 micrograms/g after four doses, while the drug was undetectable (less than 0.1 micrograms/g) in serum, spleen, liver, kidney, and brain. The half-life of elimination of AmB from the lungs was 4.8 days according to serial sacrifices done after a single dose of 3.2 mg of aero-AmB per kg. Treatment with 60 mg of aero-AmB per kg was well tolerated and produced no histopathologic changes in the lungs. The aerosol route was much more efficient than the systemic route in delivering AmB to the lungs, and it limited the accumulation of AmB in other organs. Because AmB is eliminated slowly, infrequent dosing schedules can be used. These pharmacokinetic characteristics and its proven effectiveness in an animal model make aero-AmB a highly promising new method for the prevention of pulmonary aspergillosis. Aero-AmB should also be considered for use as an adjunct to intravenous AmB for treatment of fungal pneumonias.