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.

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.

Catheter-related Malassezia furfur fungemia in immunocompromised patients.

PURPOSE, PATIENTS, AND METHODS: Malassezia furfur has usually been described as a cause of catheter-related sepsis in neonates receiving intravenous lipid emulsion. We report seven cases of catheter-related M. furfur fungemia that occurred in seven immunocompromised patients including four adults and three children who were not neonates. Only two of these patients were receiving concurrent intravenous lipid emulsion. RESULTS: All positive blood cultures were obtained from a central venous access device, one of which was a port device. Quantitative M. furfur colony counts ranged from 50 cfu/mL to greater than 1,000 cfu/mL. All seven patients were treated with amphotericin B. Blood drawn through the central lines of three patients yielded additional organisms. One central venous access device required removal due to persistently positive M. furfur blood cultures despite treatment with amphotericin B. CONCLUSION: We conclude that catheter-related M. furfur fungemia occurs in immunocompromised patients with central venous access devices whether or not they are receiving intravenous lipids. Prompt, aggressive treatment with amphotericin B (1 mg/kg/d) may spare patients removal of their central venous access device. Further studies are needed to determine the role of endogenous lipids in the development of catheter-related M. furfur fungemia and to determine if there is a seasonal incidence in populations other than neonates, since all of our cases occurred between late March and July.

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.

Sepsis due to Rhodotorula related to use of indwelling central venous catheters.

With increased use of surgically implanted silastic central venous catheters, there has been an increase in the recovery from blood cultures at Memorial Sloan-Kettering Cancer Center (New York) of environmental and skin organisms including the red yeast Rhodotorula. From 1985 through 1989, 23 patients had catheter-related Rhodotorula sepsis. All 23 patients had indwelling central venous catheters that had been in place from 1 to 22 months (average, 9.3 months) prior to the detection of fungemia. All patients had blood drawn both through the catheter and from a peripheral source, and only one patient had a peripheral blood culture positive for Rhodotorula. Colony counts of yeast from the catheter cultures often exceeded 100 (15 patients) and even 1,000 (seven patients) cfu/mL of blood. Thirteen of the patients were treated with antifungal therapy and had the catheter removed, and five patients received antifungal therapy without catheter removal (suggesting that compulsory removal of the catheter may not always be required). Five patients had the catheter removed without antifungal therapy. All patients survived the fungemic episode and experienced no recurrence of the 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.

A modified broth dilution assay for antibiotic sensitivity testing of Mycobacterium avium-intracellulare using paraffin slide cultures.

Mycobacterium avium-intracellulare (MAI) can utilize paraffin wax as the sole carbon source in basal media. Paraffin slide culture (Para SL/C) has been employed for isolation and speciation of MAI derived from clinical sources. We have evaluated an adaptation of this method for antimicrobial sensitivity testing. Sixteen clinical isolates of MAI were tested against ciprofloxacin amikacin, and azithromycin by Para SL/C and compared with sensitivities obtained with a conventional broth microtiter procedure. The system can be performed rapidly over a median time interval of 6-8 days. The MIC was defined as the lowest concentration of antimicrobial agent necessary to inhibit growth on paraffin wax coated slides. With Para SL/C, the MIC values were determined at the time when the corresponding control tubes showed confluent growth. The procedure was reproducible with all of the agents tested. The MIC50 and MIC90 values obtained from the Para SL/C assay and from serial broth microtiter dilutions correlated well for ciprofloxacin and amikacin. However, results of the MIC50 and MIC90 for azithromycin did not correlate.

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.

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.

Liver infection caused by Coniothyrium fuckelii in a patient with acute myelogenous leukemia.

A case of liver infection caused by Coniothyrium fuckelii is described in a patient with acute myelogenous leukemia. This fungus is found in the soil and can be a pathogen of plants. Coniothyrium spp. are members of the order Sphaeropsidales, an order composed of fungi whose conidiomata are usually pycnidia with the conidiogenous hymenium lining the walls of the locule. Coniothyrium spp. must be differentiated from Phoma spp. and Hendersonula spp., the two most commonly isolated members of the Sphaeropsidales.

Rapid identification using a specific DNA probe of Mycobacterium avium complex from patients with acquired immunodeficiency syndrome.

Specific DNA probes (Gen-Probe Corp., San Diego, Calif.) for Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium tuberculosis were compared with conventional methods for the identification of isolates of the Mycobacterium avium complex. A total of 56 isolates of M. avium complex were recovered from 34 respiratory, 13 blood, 6 stool, and 3 urine samples from 23 patients. A total of 33 isolates were tested directly from Middlebrook 7H11 agar plates, and 23 isolates were tested directly from BACTEC radiometric 12B bottles (Johnston Laboratories, Inc., Towson, Md.). Of the 56 M. avium complex isolates, 41 tested positive with the M. avium probe, 4 were positive with the M. intracellulare probe, and 7 were positive with both probes. Four direct tests from BACTEC bottles were initially negative but were subsequently M. avium probe positive when subcultures from Lowenstein-Jensen agar were tested. All 56 strains were negative when tested with the M. tuberculosis probe.

Evaluation of the Quantum II yeast identification system.

We compared three methods for identifying clinical yeast isolates: Abbott Quantum II, API 20C, and a modified BBL Minitek system. The API 20C and modified Minitek systems agreed on the identification of 243 of 245 yeasts (99.2%). The Quantum II system correctly identified 197 (80.4%), incorrectly identified 19 (7.8%), and did not identify 29 (11.8%) of the yeasts. Most of the misidentifications with the Quantum II occurred because assimilation or biochemical results were false-positive. Sixteen different species of yeasts and 16 different Quantum II substrates contributed to the discrepancies. On retesting with the Quantum II, 31% of the discrepant strains were correctly identified, while the remaining 69% were incorrectly identified or were not identified. Erroneous biochemical and assimilation results were also noted with yeasts that were correctly identified by the Quantum II system.

Catheter-associated fungemia caused by Fusarium chlamydosporum in a patient with lymphocytic lymphoma.

A case of catheter-associated fungemia caused by Fusarium chlamydosporum is described in a patient with lymphocytic lymphoma. The fungus, which has been isolated from soil but not reported to cause human infection, characteristically produces microconidiophores that are polyphialides bearing microconidia that are spindle-shaped but never globose. Results of in vitro antimicrobial susceptibility tests depended on the test conditions used.

Infections caused by Mycobacterium avium complex in immunocompromised patients: diagnosis by blood culture and fecal examination, antimicrobial susceptibility tests, and morphological and seroagglutination characteristics.

The Mycobacterium avium complex, only rarely described as an invasive pathogen in humans, has recently been reported to frequently cause disseminated disease in patients with the acquired immune deficiency syndrome. Between February 1981 and February 1984 at Memorial Sloan-Kettering Cancer Center, 30 patients with acquired immune deficiency syndrome, 3 patients with leukemia, and 2 patients with congenital severe combined immunodeficiency syndrome developed disseminated M. avium complex infection. Mycobacteria were often found in multiple sites both antemortem and postmortem. Blood cultures were a reliable method for detecting disseminated infection, and the new lysis blood culture systems provided an efficient technique for determining the number of organisms per milliliter of blood. Acid-fast stains and cultures of fecal specimens were also helpful in diagnosing infection. Most of the mycobacteria were serovar 4 (77%), and most (86%) produced a deep yellow pigment. All isolates were susceptible to standard concentrations of clofazimine, cycloserine, and ansamycin, but tended to be resistant to isoniazid, streptomycin, ethambutol, ethionamide, and rifampin.

Continuous high-grade mycobacterium avium-intracellulare bacteremia in patients with the acquired immune deficiency syndrome.

Serial quantitative blood cultures were performed before and during treatment in four patients with the acquired immune deficiency syndrome (AIDS) and Mycobacterium avium-intracellulare bacteremia. Initial colony counts were 350 to 28,000 cfu/ml, the counts declined substantially with treatment in two patients, and they declined modestly with treatment but rose when it was stopped in the other two. In one patient who was studied in detail, most of the circulating organisms were within the leukocytes, colony counts in blood subjected to lytic agents were 1.9- to 5.2-fold higher than in unlysed blood, and there were 10(5) to 10(6) times more organisms per gram in several tissue specimens obtained at autopsy than per milliliter of blood. It is concluded that continuous high-grade bacteremia is common in patients with AIDS and severe M. avium-intracellulare infections and that serial quantitative blood cultures provide a potential means for studying treatment in these patients.

Routine aerobic terminal subculturing of blood cultures in a cancer hospital.

Routine terminal aerobic subcultures of macroscopically negative blood culture bottles were evaluated during a 15-month period when 30,000 blood cultures were processed. Each blood culture set consisted of a vented and an unvented 50-ml broth bottle. Forty-eight pathogens and 47 contaminants were isolated only from terminal subcultures. Twenty-two of the significant isolates were yeasts (usually recovered from vented bottles), and 10 were Pseudomonas aeruginosa (usually recovered from unvented bottles). Blood cultures that were positive by terminal subculture provided clinically relevant information in many cases, whether other blood cultures were positive or not. Microbiology laboratories, particularly those in hospitals where yeasts and P. aeruginosa are commonly isolated from blood specimens, should evaluate carefully the need for terminal subcultures of blood culture bottles before abandoning their use.

Comparative recovery of bacteria and yeasts from lysis-centrifugation and a conventional blood culture system.

Blood cultures obtained with a lysis-centrifugation (L-C) system and a conventional two-bottle broth system were compared for the recovery of bacteria and yeasts from 7,000 cultures. The L-C system recovered significantly more total organisms, Escherichia coli, and Candida spp. and detected more patients with bacteremia and fungemia due to members of the family Enterobacteriaceae and yeasts. The broth system recovered significantly more streptococci and detected significantly more low-level Pseudomonas bacteremias. Polymicrobic bacteremia and fungemia were detected equally well by either culture system. Aerobic organisms grew equally well on blood, chocolate, or brain heart infusion agar plates used for L-C inoculation. A total of 82% of colony counts measured no more than 10 CFU/ml of blood, and it was at these low levels that enhanced detection of organisms by either system was observed. The L-C system isolated organisms and detected yeasts more rapidly than did the broth system. Contaminants occurred in 8.2% of L-C cultures and 1.9% of broth cultures. Low colony counts on L-C plates occurred for both Staphylococcus epidermidis contamination and septicemia.

The prevalence of yeasts in clinical specimens from cancer patients.

Yeasts recovered from cancer patients during a 15-month period were speciated, and the prevalence of these isolates in various types of clinical specimens was determined. Five species, including Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, and Torulopsis glabrata, accounted for 97.1% of the isolates. Eighteen different species were recovered. Respiratory and urine specimens yielded 75% of the organisms. C. albicans, C. tropicalis, and C. parapsilosis were recovered in about equal frequency from blood cultures. Certain species usually were recovered from one type of specimen: Candida quilliermondii from urine, Trichosporon cutaneum and Candida pseudotropicalis from respiratory sites, and Cryptococcus neoformans from spinal fluid. Pityrosporum orbiculare was isolated only from ear and urine cultures. Most of the yeasts (95.4%) were identified within 48 hours after isolation.

Nitrogen-deficient medium in the differential isolation of Klebsiella and Enterobacter from feces.

On a nitrogen-deficient agar medium, the tribe Klebsielleae formed large, glistening, mucoid colonies which were easily distinguished from other colony types. Of 113 Klebsielleae isolates from human feces which were characterized, Klebsiella accounted for 88% of the total; 75% were K. pneumoniae; K. ozaenae (13%) was isolated from one individual only. The remaining strains (12%) were identified as Enterobacter cloacae. Counts (for the tribe) ranged from 10(2) to 10(6), with a median of 10(4); 9 of 53 stool specimens were negative. K. pneumoniae was also isolated from 6 of 41 frozen foil-pack foods. Anaerobic studies at room temperature and 37 C revealed no appreciable differences from aerobic plates. The nitrogen-deficient medium appeared better than E M B for isolation of Klebsielleae when they were present in low numbers relative to other coliforms; slime production by Klebsielleae concomitant with minimal growth of other bacteria is involved.