1995, 33(12):3290. J. Clin. Microbiol. 

Rodriguez-TudelaPorto, M Gonzalez, J V Martinez-Suarez and J L M Alvarez, B Lopez Ponga, C Rayon, J Garcia Gala, M C Roson leukemic patients.prolificans (inflatum): four fatal cases in Nosocomial outbreak caused by Scedosporium

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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1995, p. 3290–3295 Vol. 33, No. 120095-1137/95/$04.0010Copyright q 1995, American Society for Microbiology

Nosocomial Outbreak Caused by Scedosporiumprolificans (inflatum): Four FatalCases in Leukemic Patients

MARIA ALVAREZ,1* BEATRIZ LOPEZ PONGA,1 CONSUELO RAYON,2

JOSE GARCIA GALA,2 MARIA CONSOLACION ROSON PORTO,2

MARTA GONZALEZ,3 JOAQUIN V. MARTINEZ-SUAREZ,4

AND JUAN LUIS RODRIGUEZ-TUDELA4

Servicio de Microbiologıa,1 Servicio de Hematologıa,2 and Servicio de Anatomıa Patologica,3

Hospital Central de Asturias, Oviedo, and Unidad de Micologıa, Centro Nacional deMicrobiologia, Instituto de Salud Carlos III, Majadahonda, Madrid,4 Spain

Received 27 February 1995/Returned for modification 3 April 1995/Accepted 11 September 1995

Four cases of fatal disseminated Scedosporium prolificans (inflatum) infection occurring in neutropenicpatients are reported. Because of hospital renovation, the patients were cared for in a temporary hematologicfacility. S. prolificans (inflatum) was isolated from blood cultures of these four patients, two of whom underwentfull necropsy, and revealed abundant vegetative hyphae and ovoid conidia with truncate bases in many organs.In vitro susceptibility testing of fungal strains showed all isolates to be resistant to amphotericin B, flucytosine,miconazole, ketoconazole, fluconazole, and itraconazole, with MICs greater than 16 mg/ml. The reportedinfections, two in each of two rooms, occurred over a period of 1 month, with very similar clinical outcomes.Circumstancial evidence suggested a nosocomial outbreak, but the environmental samples collected from therooms, corridors, and adjacent areas did not yield S. prolificans (inflatum). Nevertheless, circumstantialevidence suggested a nosocomial outbreak of S. prolificans (inflatum) infection.

Since the description of Scedosporium inflatum (Malloch andSalkin) as a human pathogen (14), investigators have suggestedthat the appropriate species epithet is Scedosporium prolificans(8, 11) reports of several cases have enlarged the clinical spec-trum of this fungus (22, 29, 32, 33). Furthermore, it seems thatS. prolificans causes a spectrum of clinical illness similar to thatproduced by Scedosporium apiospermum (30) but broader thanwas previously thought, especially among immunocompro-mised patients (5, 7, 10, 13, 26, 28).The natural habitat of S. prolificans remains unknown, but it

has been isolated from flower pots and is most likely a soilsaprobe (6, 27).Outbreaks of nosocomial infections caused by contaminat-

ing environmental fungi when the number of spores are in-creased by activities such as nearby building construction orthe presence of contaminated air-conditioning systems (11, 12,31). A nosocomial outbreak involving four cases of fatal dis-seminated infection by S. prolificans in leukemic patients isreported. The four cases occurred sequentially during recon-struction work at the hospital, over the short period of 28 daysin two rooms in the provisional hematology unit.The provisional hematology unit was located on the fifth

floor of the hospital in an ordinary hospitalization unit, andtwo single rooms were designated for the isolation of immu-nocompromised patients. These two rooms were at the entry tothe unit, opposite the construction work on the same floor. Thebarrier between the construction work and the unit was twosets of double doors, and the distance was 20 m. There was no

trafficking of construction workers between the constructionarea and the provisional unit.

MATERIALS AND METHODS

Case identification. Between 26 November and 14 December 1992, four casesof disseminated S. prolificans infection were identified in the hematology depart-ment of Hospital Central de Asturias, a large, tertiary care community teachinghospital, built 30 years ago. Hospital renovation was being carried out, and theHematology Department was provisionally housed in a nonprotected area (or-dinary single rooms). The patients received treatment in private rooms of theinverse isolation class (mask and hand washing required). See Fig. 1 for acomparison of the clinical outcomes.Clinical samples. Samples of urine, exudate, and feces, etc., were processed in

the microbiology laboratory following standard methods in order to detect thepresence of aerobic bacteria, anaerobic bacteria, and fungi (2).Blood samples (10 ml) were inoculated into aerobic and anaerobic bottles

(Bactec; BBL). They were incubated at 378C, and daily readings with a BactecNR-730 automatic detector (Beckton Dickinson, Microbiological Systems, Cock-eysville, Md.) were made until the detection of growth. If after 8 days the cultureswere still negative, the samples were subcultured on plates of chocolate agar.These plates were then incubated at 378C in anaerobic jars (Oxoid) at 10% CO2for 48 h.When fungal infection was suspected, the blood sample (10 ml) was also

processed by using a lysis-centrifugation technique (Isolator 10; Merck) by sub-culturing the sediment on plates of Sabouraud dextrose agar (SDA), sheep bloodagar, and chocolate agar; the plates were incubated at 378C for 8 days, and thesamples were also subcultured in a biphasic medium (HEMOLINE; bioMerieux)and incubated at 308C for 40 days.Environmental surveillance. Saline-moistened sterile cotton swabs were used

for collecting dust samples from the floors, ceilings, and walls of the rooms, thecorridors, and the nurses’ station in the affected unit and reconstruction areas.The swabs were inoculated onto SDA without and with amphotericin B at a finalconcentration of 10 mg/ml. The plates were incubated at 308C for 10 days. No airsamples were taken.Dust samples were taken several times: the same day that the first blood

culture fungus growth was identified, simultaneously with the fourth death, thenext day, and during the following month.Mycology. Each fungal colony detected was subcultured on two plates of SDA

(Difco), Mycosel agar (BBL), malt extract agar (Oxoid), and potato dextroseagar (Merck), and the two plates of each set were incubated for 2 weeks at 30 and

* Corresponding author. Mailing address: Servicio de Microbiolo-gia, Hospital Central, S/S Edificio Materno-Infantıl, C/ Celestino Vil-lamıl s/n, 33006 Oviedo, Spain. Phone: 8-5108000, ext. 38215. Fax:8-5108015.

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378C, respectively. The microscopic examination of the isolates was carried outby using a previously described technique (19).Antifungal susceptibility testing. Susceptibility testing was performed accord-

ing to the broth dilution micromethod described by Schmitt et al., with minormodifications (24).The six antifungal drugs used in the susceptibility testing procedure were

amphotericin B (Squibb), flucytosine (Hoffman-La Roche Laboratories), keto-conazole and itraconazole (Janssen Pharmaceutica), fluconazole (Pfizer), andmiconazole (Sigma). Stock solutions of ketoconazole, fluconazole, and micon-azole were prepared at 12.8 mg/ml in 100% dimethyl sulfoxide. Amphotericin Band itraconazole (1.6 mg/ml) were solubilized in 100% dimethyl sulfoxide, andflucytosine (1.6 mg/ml) was dissolved in sterile distilled water. All these solutionswere stored at 2708C.The medium used for susceptibility testing was RPMI 1640 with L-glutamine

(Gibco) buffered with morpholinepropanesulfonic acid (MOPS) buffer (Sig-ma) to a final molarity of 0.165, adjusted to pH 7.0 by using 10 M NaOH (25)and supplemented with 18 g of glucose (RPMI–2% glucose) per liter (20).We employed RPMI–2% glucose as a testing medium because it providesgood cell growth, the end points are also more easily read, and it is both easyto prepare and economical and does not contain antagonists for flucytosineactivity.Sterile plastic microtitration plates containing 96 round-bottom wells each,

with their corresponding covers (Greiner), were employed. The concentrationsof ketoconazole, fluconazole, and miconazole ranged from 128.0 to 0.25 mg/ml;itraconazole, amphotericin B, and flucytosine were used at concentrations of 16.0to 0.03 mg/ml. The plates contained 10 twofold serial dilutions of each antifungalagent in each row and a drug-free medium in wells 11 and 12 for sterility checkand growth control, respectively.Homogeneous inoculum suspensions were prepared from SDA (Oxoid) cul-

tures grown at 288C for 7 days, according to the method of Espinel-Ingroff andKerkering (4). Briefly, suspensions were made by rubbing the surface of SDAslants with a loop after the addition of sterile water, washing the scapings, andthen transferring the suspensions to a sterile tube. After the heavy particles of thesuspensions were allowed to settle, the upper homogeneous suspensions wereadjusted with a spectrophotometer set at 530 nm to 78 to 82% transmission toobtain final suspensions containing 1 3 106 to 5 3 106 CFU/ml. Colony countson the SDA plates were determined. The suspensions were diluted 1:10 withRPMI–2% glucose, and 10 ml was dispensed in each of the wells (ca. 104

CFU/ml).After inoculation, the microtiter plates were incubated at 358C for 48 h in a

humid atmosphere. The plates were then read macroscopically with a mirror, andthe MIC was defined as the lowest concentration of the antifungal agent thatcompletely inhibited fungal growth.Aspergillus fumigatus ATCC 9197 and Aspergillus versicolor ATCC 11730 were

used as controls in all plates. For comparative purposes, one strain of S. apio-spermum from our collection was also included.Thus, because of the scarcity of reported cases of systemic infections by S.

prolificans, we have deposited an isolate from each of the patients in the Unidadde Micologıa, Centro Nacional de Microbiologıa, Instituto de Salud Carlos III,Majadahonda Madrid, Spain (no. M93/1, M93/2, M93/3, and M93/4), and thefirst of these was also deposited in the Mycological Reference Laboratory of thePublic Health Laboratory Services, National Collection of Pathogenic Fungi,London, England (no. 7.108).

Autopsy. A complete macroscopic and microscopic study was performedfor two cases, including lungs, liver, kidneys, spleen, thyroid glands, andlymph nodes. The histological sections were placed in 10% Formol and thenstained with hematoxylin-eosine, periodic acid-Schiff stain, and methenamine-silver.

CASE REPORTS

Case 1. A 27-year-old woman was admitted with acute my-eloid leukemia. On the 12th day after admission she becamefebrile, and a treatment of ceftazidime plus amikacin wasstarted. On the 19th day, she became febrile again, and a chestradiograph revealed perihilar infiltrate. Amphotericin B (1mg/kg of body weight per day) plus imipenem was started, buther condition worsened. She died 31 days after admission. Twoblood cultures (Bactec) obtained on this day were positive forS. prolificans.An autopsy was performed. Microscopic study revealed ex-

tensive and hemorrhagic necrotic zones with thrombosed ves-sels. Hyphae branched at acute angles and were accompaniedby rounded, small elements observed around and within vesselsas well as in the lung parenchyma. These same structures werealso visualized in the liver, suprarenals gland, kidneys, andspleen. Samples for culturing were not taken.Case 2. A 45-year-old woman was admitted with acute my-

eloid leukemia. On the seventh day after admission, she had afever and a neutrophilic count of ,100 3 109/liter. Ceftazi-dime plus amikacin treatment was started. After 48 h of treat-ment, she continued to be febrile, and the antibiotic treatmentwas changed to vancomycin plus aztreonam. On the 12th day,the patient’s condition worsened, and she reported thoracicpain characteristic of pneumonia. Amphotericin B (1 mg/kg/day) plus imipenem was started. An X-ray of the thoraxshowed diffuse pulmonary infiltrates bilaterally. The patientdied 22 days after admission. Blood cultures obtained 48 h(three Isolator cultures) and 24 h (three Bactec cultures) be-fore death were positive for S. prolificans. A postmortem ex-amination was not authorized.Case 3. A 79-year-old woman was admitted with acute lym-

phoblastic leukemia. After 1 week she became febrile. Sheresponded well to treatment with ceftazidime plus amikacin,but on the 12th day she became febrile again. The neutrophilic

FIG. 1. Comparison of the four cases of S. prolificans infection. AD, admission date; F, fever; AMB, amphotericin B treatment; EXT, date of death.

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count was ,100 3 109/liter. A chest radiograph revealed bi-lateral pulmonary infiltrate.On the 25th day, her condition worsened. A chest X-ray

revealed right pleural effusion, and treatment with amphoter-icin B (1 mg/kg/day) plus imipenem was begun. The patientdied 27 days after admission. Two blood cultures (Bactec)taken 48 h before death were positive for S. prolificans. Apostmortem examination was not authorized.Case 4. A 54-year-old woman was admitted because of an

acute myeloid leukemia. Chemotherapy with adriamycin plus1-b-D-arabinofuranosyl cytosine was started. On the 8th dayafter admission, she had a fever of 388C, treatment with cefta-zidime plus amikacin was begun, and teicoplanin was addedafter 48 h. During the third week after admission, she becamefebrile again, the neutrophilic count was ,100 3 109/liter, andtreatment with amphotericin B (1 mg/kg/day) plus imipenemwas started.Three days later, the patient’s condition worsened, with bi-

lateral ocular and muscular pain. An X-ray film of the chestshowed a bilateral pulmonary infiltrate. The results of a com-puted tomography scan of the head were normal. The patientdeveloped acute respiratory and renal failure and died 23days after admission. A blood culture (Bactec), tracheal aspi-rate, and another blood culture (Isolator) obtained 96, 72,and 24 h, respectively, before death were positive for S. pro-lificans.An autopsy was performed. The histopathological findings

revealed that the lungs had increased in weight. The paren-chyma was solid, showing pinkish-to-red rounded areas. Mi-croscopically, bronchopneumonic zones with little cellular re-

action, alveolar masses of fibrinoid material, and extensiveareas of thrombotic vessels and necrosis were observed (Fig.2). In all these zones, spreading myriad mycotic structuresformed by long hyphae, which branched at acute angles, androunded structures with small sizes were seen (Fig. 3). Thisfungal invasion was also observed in the liver, spleen, kidneys(Fig. 4), thyroid glands, and lymph nodes. Samples for cultur-ing were not taken.

RESULTS

S. prolificans grew on chocolate agar after routine subcul-turing of apparently fungus-negative samples in Bactec bottles.The time required to detect S. prolificans in blood culture(Bactec) always exceeded 7 days: 15, 10, 10, and 9 days, re-spectively, for the four cases, but with the Isolator, the timeneeded was 48 and 72 h in cases 2 and 4, respectively.On subculture, the fungi grew rapidly on SDA, malt extract

agar, and potato dextrose agar. The colony was flat, moist,spreading, olive-grey to black in color, and arachnoid to woollyin appearance. With age, the four isolates developed sectionsthat were woolly in appearance (Fig. 5).Microscopic examination revealed ovoid, smooth, one-celled,

thin-walled conidia. The conidiogenous cells were annellides,which occurred isolated along the hyphae and in clusters of2 to 5 at the apex of unbranched conidiophores. The conid-iogenous cell showed the distinctive swollen base of S. prolifi-cans. None of the four isolates grew in cycloheximide-contain-ing media. The overall appearance of the colony and the

FIG. 2. Lung. Methenamine-silver staining of lung tissue shows a thrombosed vessel by the fungal mass, with wall and lumen invasion. Magnification, 3100.

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characteristic microscopic morphology were consistent withthose of S. prolificans, as shown in previous reports (3, 7, 9, 10,14, 15, 21, 22, 33).The activities of the six antifungal drugs tested against the

four isolates of S. prolificans and control strains are shown inTable 1.S. prolificans failed to grow in any of the environmental

samples processed for 2 months, although there was massivegrowth of other environmental fungi, such as Aspergillus fu-migatus Penicillium sp., Rhizomucor sp., and, to a lesser extent,Alternaria sp. and Cladosporium sp. All dematiaceous funguswas subcultivated for identification.

DISCUSSION

Fungal infections are becoming an increasingly significantproblem and are a major cause of mortality in immunocom-promised patients. Although the major risk factor is granulo-cytopenia, other factors such as multiple antibiotic therapy,humoral and cellular immunodeficiencies, surgery, and in-dwelling catheters are related with these infections.Several cases of disseminated infection caused by S. prolifi-

cans have been reported. The affected populations were pre-dominantly immunosuppressed patients (5, 7, 18, 26, 28, 33)although there are two reported cases of endocarditis (5, 8) innonimmunosuppressed patients. The clinical outcome is gen-erally fatal. In Spain, eight cases of disseminated infectioncaused by S. prolificans in leukemic patients have been re-ported (7, 10, 16, 21, 28), all of which occurred in an isolatedway. Death was the usual outcome, despite antifungal treat-ment.

To our knowledge, this is the first report of disseminatedS. prolificans infections occurring as a nosocomial outbreakassociated with renovation inside the hospital. At the time, thepatients were cared for in a provisional unit without the stan-dard protective measures for granulocytopenic patients; theywere located in single rooms with restricted entry and obliga-tory use of a mask, wedge, and dressing gown. There were noair-conditioning or potted plants in this zone, and the unit was,in fact, closed off from the renovation zone. The four casesoccurred in two adjacent rooms which were occupied succes-sively by these four patients for 28 days.When the fourth death occurred, simultaneously with the

fungal identification of the positive blood cultures from thefirst two deceased patients, the two rooms were temporarilysealed and dust samples were processed several times. Thehematology unit was moved to a different area (from the fifthfloor to the second floor), until the permanent hematologicunit was completed, and no additional cases have been de-tected since.The clinical symptoms and the radiological findings of X-ray

of the thoraxes of the four patients suggested aerial transmis-sion. Many investigators emphasize the importance of isolationduring granulocytopenia in order to avoid disseminated myco-ses (1, 16). However, the environmental samples taken werenegative despite the fact that a selective medium for S. prolifi-cans was used (SDA plus amphotericin B). Therefore, thesource of this nosocomial outbreak and the transmission routeof the fungus remain unknown.The isolation of the etiological agent was closely premortem,

as the blood cultures taken on the day of or 48 and 72 h beforedeath were positive. Furthermore, only the lysis centrifugation

FIG. 3. Lung. Methenamine-silver staining shows fungal filaments and a detail of the ramifications. Magnification, 3400.

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method provided fungal growth within 48 to 72 h, as in cases 2and 4, respectively. Fungi grew in the subcultures made fromBactec blood culture bottles after 7 days of incubation. After 7days, Bactec blood culture bottles were subcultured in a blindfashion, and at this time the fungus was detected. Unfortu-nately, the identification of the fungus was made only after thepatients’ deaths. In our laboratory, only two previous culturesof a Scedosporium sp. from clinical samples in the last 15 yearswere available, and they were S. apiospermum.In cases when a biopsy can be performed, the observation of

vegetative hyphae together with ovoid conidia in histologicalsections could suggest S. prolificans infection, (15, 21, 32), andour experienced pathologist had never before seen these ele-ments. Fungal culture is always necessary for correct identifi-cation.Even though an early diagnosis was reached, the therapeutic

outcomes were disappointing. In previous investigations,Salkin et al. (22) found four isolates for which the MICs of

FIG. 4. Kidney. Periodic acid-Schiff staining of kidney tissue shows fungal invasion of interstitial tissue, with no glomerular involvement. Magnification, 3100.

FIG. 5. Growth of fungus on potato dextrose agar after 14 days.

TABLE 1. Antifungal MICs of S. prolificans

FungusNo. ofsamplestested

MIC (mg/ml)a

AB F KZ FZ IZ MZ

S. prolificans 4 .16 .16 128 .128 .16 128S. apiospermum 1 4 .16 2 .128 1 0.5A. fumigatus 1 0.5 .16 8 .128 0.5 4A. versicolor 1 2 4 2 .128 0.5 2

a AB, amphotericin B; F, flucytosine; KZ, ketoconazole; FZ, fluconazole; IZ,itraconazole; MZ, miconazole.

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itraconazole were lower than 0.018 mg/ml, and Wood et al. (33)found that one isolate of S. prolificans was susceptible to itra-conazole at 25 mg/ml, with clinical response at high doses, butthis was not seen in our case. Our data also confirm previouslypublished results (21, 22) regarding the resistance of S. prolifi-cans to amphotericin B, ketoconazole, and miconazole, com-pared with the lower MICs for S. apiospermum. Antifungalsusceptibility tests for filamentous fungi remain unstandard-ized, and the in vitro results cannot be extrapolated to the invivo outcome. However, many investigators agree that S. pro-lificans shows a high degree of resistance to antifungal drugsirrespective of the method of detection used.Wood et al. (33) have reported the only case of an immu-

nosuppressed patient without fatal outcome who responded toantifungal drugs plus granulocyte colony-stimulating factor.Whether that treatment can be successful remains unan-swered, and further studies are needed (17, 23). At the presenttime antifungal treatment cannot be expected to play a majorrole in recovery from S. prolificans infections.

ACKNOWLEDGMENTS

We are grateful to Colin R. Cambell of the Mycological ReferenceLaboratory, Public Health Laboratory Service, London, England, forcollaboration and to David H. Wallace for the English language revi-sion of the manuscript. Also, we are grateful to both peer reviewers,who spent their time in correcting the manuscript, for their recom-mendations that improved this report.

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