Clinical, pathologic and epidemiologic features of infection with Scedosporium prolificans: four cases and review

Objective: To describe the features of infection with Scedosporium prolificans and to investigate the possibility of a common source of infection.
Methods: S.prolificans caused invasive infections in four patients at Liverpool Hospital from 1994 to 1997, prompting a pathologic and epidemiologic investigation. Blood cultures were processed by either the BACTEC NR660 or VITAL systems. MIC testing was performed with Etest strips. Strain differentiation of isolates from three of the patients and two soil samples from the home of one of the patients was performed by allozyme electrophoresis.
Results: The four cases represented disseminated infections that arose during prolonged neutropenia, and progressed relentlessly despite treatment, including in one case high-dose liposomal amphotericin B. In two cases, VITAL blood culture bottles contained mycelia of S. prolificans without the reader having signaled. An autopsy was performed in three of the cases. Angio-invasion, tissue necrosis and multiple abscesses were found in each patient. Multiple air samples from the ward were negative for S. prolificans . The organism was grown from two samples of pot plant soil from the home of one patient. Allozyme electrophoresis performed on isolates from three cases and pot plant soil indicated that all strains were unrelated. Furthermore, two patients appeared to harbor at least two different strains of S. prolificans .
Conclusions: S. prolificans causes disseminated infection in neutropenic patients. Antifungal treatment, including high-dose liposomal amphotericin B, is ineffective in overcoming these infections. Genetic techniques discriminated all strains and suggested that an outbreak had not occurred. Multiple strains of S. prolificans were isolated from two patients, indicating the possibility of mixed infections occurring.     

Characterization of Scedosporium prolificans clinical isolates by randomly amplified polymorphic DNA analysis.

Fingerprinting by randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate Scedosporium prolificans isolates. A total of 59 arbitrary primers were screened with six unrelated S. prolificans isolates, and a panel of 12 primers was selected. The 12 primers were then used to detect DNA polymorphisms among 17 S. prolificans isolates from 11 patients with systemic S. prolificans infections diagnosed in three hospitals located in geographically different areas of Spain. Eight patients were diagnosed with S. prolificans infection in a single institution over a 6-year period, and two other patients were diagnosed with S. prolificans infection in a different hospital over a 1-year period. No single primer allowed for the discrimination of all the isolates from different patients, but this was possible by combining the RAPD patterns from three primers (UBC 701, AB1.08, and AB1.11 or UBC 701, AB1.08, and UBC 707). However, multiple isolates from the same patient were identical. In this study, we also compared a visual method and a computerized method for the analysis of the RAPD patterns. Both methods were satisfactory and gave few discordances, but given the advantages and disadvantages of each method, both systems should be used together. RAPD analysis provided a fast and economical means of typing S. prolificans isolates, with a high level of discrimination among unrelated isolates. Typing by RAPD analysis confirmed that the S. prolificans infections were epidemiologically unrelated.     

Ribosomal DNA internal transcribed spacer analysis supports synonomy of Scedosporium inflatum and Lomentospora prolificans.

Scedosporium inflatum is a dematiaceous opportunistic pathogen originally described by D. Malloch and I.F. Salkin (Mycotaxon 21:247-255, 1984). However, E. Gueho and G. S. De Hoog (J. Mycol. Med. 118:3-9, 1991) recently suggested reducing this mold to synonomy with Lomentospora prolificans on the basis of their similar morphological and molecular characteristics. We have investigated the ribosomal DNA internal transcribed spacers (ITS), i.e., ITS I and ITS II, of 18 isolates, including these two fungi and a closely related pathogen, Scedosporium apiospermum, and its telemorph, Pseudallescheria boydii. Identical ITS restriction fragment length polymorphisms were found in eight isolates of S. inflatum and L. prolificans. These results support Gueho and De Hoog's proposal to combine S. inflatum and L. prolificans into the binomial Scedosporium prolificans. However, the ITS I sequence of S. apiospermum and the ITS restriction fragment length polymorphisms of S. apiospermum and P. boydii were found to be significantly different from those of S. inflatum and L. prolificans. The ITS restriction pattern differences may be valuable in clinical settings for distinguishing these fungi.     

Comparison of the virulence of Scedosporium prolificans strains from different origins in a murine model

Scedosporium prolificans is an emerging opportunist fungus that causes different types of infections in immunocompetent and immunosuppressed people. These infections show an irregular geographical distribution and, generally, disseminated systemic infections are noticed only in specific countries. This study used a murine model of disseminated infection by this fungus to assess if strains from different origins have different virulence. Two strains from each of four different sources (disseminated infection, localised infection, asymptomatic cystic fibrosis patients and the environment) were tested. Two strains of S. apiospermum of clinical origin were also included in the study; these were clearly less virulent than those of S. prolificans. The S. prolificans strains tested were classified in three groups according to their virulence. The groups with higher and lower virulence were represented by only one strain each, and the intermediate group contained six strains. No significant differences were found between the strains from different geographic areas or different forms of disease.     

Nosocomial outbreak caused by Scedosporium prolificans (inflatum): four fatal cases in leukemic patients.

Four cases of fatal disseminated Scedosporium prolificans (inflatum) infection occurring in neutropenic patients are reported. Because of hospital renovation, the patients were cared for in a temporary hematologic facility. S. prolificans (inflatum) was isolated from blood cultures of these four patients, two of whom underwent full necropsy, and revealed abundant vegetative hyphae and ovoid conida 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 micrograms/ml. The reported infections, 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 the rooms, corridors, and adjacent areas did not yield S. prolificans (inflatum). Nevertheless, circumstantial evidence suggested a nosocomial outbreak of S. prolificans (inflatum) infection.     

Fungemia due to Scedosporium prolificans: a description of two cases with fatal outcome

Two cases, probably related, of fungemia due to Scedosporium prolificans are described in two patients with acute leukemia. Both were admitted to the hematological ward in nearby rooms, during building work in the hospital. After a previous bacterial sepsis in the neutropenic phase, which improved with antibiotic treatment, the respiratory status in both patients deteriorated presenting acute dypsnea, with a lung infiltrate in one of them. A few hours later both patients died. Blood cultures were positive for S. prolificans . These two new cases of S. prolificans infection stress the importance of awareness of this emerging pathogen in patients who suffer a hematologic malignancy during the neutropenic phase, especially if building work is taking place in the hospital.     

Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.

Chronic respiratory tract infections caused by Staphylococcus aureus are common in patients with cystic fibrosis (CF). Recently, it was shown in a few CF patients that S. aureus isolates produce capsular polysaccharides (CPs). However, it is not known whether this is a common feature and whether an immune response to CPs in CF is detectable. Therefore, we examined 170 S. aureus isolates from CF patients and healthy individuals for production of CP types 5 and 8 by using monoclonal antibodies. We found that CP-producing staphylococcal isolates were randomly distributed among CF patients and healthy carriers. Eighty-five percent of all isolates produced CPs, 77% of which were type 8. Examination of one sputum sample by an immunofluorescence technique suggested that production of CPs is not an in vitro phenomenon. S. aureus isolates from various sites of a single person often yielded more than one CP type. A random distribution of S. aureus strains with CP type 5 or 8 from the skin and respiratory tracts of patients and from the skin of healthy individuals was found. Antibody response to CP types 5 and 8, measured by enzyme-linked immunosorbent assay, was not elevated in CF patients with chronic S. aureus lung infection in comparison with healthy carriers. On the contrary, in CF patients the ratios of antibodies to CP 8 were significantly lower (P less than 0.005; alpha = 0.025). The ratios of antibodies to CP types did not change when monitored longitudinally over several months. This study suggests that the production of CPs is a universal property of S. aureus and that infected CF patients do not have elevated ratios of antibodies to these antigens.     

Human phagocytic cell responses to Scedosporium prolificans.

Scedosporium prolificans (SP) is an emerging opportunistic dematiaceous mould that causes serious infections in immunocompromised patients. Antifungal activities of human polymorphonuclear (PMN) and mononuclear (MNC) leukocytes against five SP isolates and Aspergillus fumigatus (AF) were evaluated. While monocyte-derived macrophages (MDM) phagocytosed conidia of both organisms comparably, they inhibited the germination of S. prolificans conidia less efficiently than those of A. fumigatus. Unopsonized hyphae of SP strains decreased the superoxide anion (O2-) produced by both PMN and MNC, whereas opsonized hyphae significantly stimulated it. In comparison to AF, phagocytes generally exhibited equal oxidative burst in response to SP. While PMN- and MNC-induced hyphal damage was similar among SP strains, phagocytes tended to damage SP hyphae to an equal or higher degree than AF hyphae. The susceptibility of SP to phagocytes contrasts with its high resistance to antifungal agents and may be related with the very low pathogenicity of the mould.     

Use of turbidimetric growth curves for early determination of antifungal drug resistance of filamentous fungi

A previously described microbroth kinetic system (J. Meletiadis, J. F. Meis, J. W. Mouton, and P. E. Verweij, J. Clin. Microbiol. 39:478-484, 2001) based on continuous monitoring of changes in the optical density of fungal growth was used to describe turbidimetric growth curves of different filamentous fungi in the presence of increasing concentrations of antifungal drugs. Therefore, 24 clinical mold isolates, including Rhizopus oryzae, Aspergillus fumigatus, Aspergillus flavus, and Scedosporium prolificans, were tested against itraconazole, terbinafine, and amphotericin B according to NCCLS guidelines. Among various parameters of the growth curves, the duration of the lag phase was strongly affected by the presence of antifungal drugs. Exposure to increasing drug concentrations resulted in prolonged lag phases of the turbidimetric growth curves. The lag phases of the growth curves at drug concentrations which resulted in more than 50% growth (for itraconazole and terbinafine) and more than 75% growth (for amphotericin B) after 24 h of incubation for R. oryzae, 48 h for Aspergillus spp., and 72 h for S. prolificans were 4 h longer than the lag phases of the growth curves at the corresponding drug-free growth controls which varied from 4.4 h for R. oryzae, 6.5 h for A. flavus, 7.9 h for A. fumigatus, and 11.6 h for S. prolificans. The duration of the lag phases showed small experimental and interstrain variability, with differences of less than 2 h in most of the cases. Using this system, itraconazole and terbinafine resistance (presence of >50% growth) as well as amphotericin B resistance (presence of >75% growth) was determined within incubation periods of 5.0 to 7.7 h for R. oryzae (for amphotericin B resistance incubation for up to 12 h was required), 8.8 to 11.4 h for A. fumigatus, 6.7 to 8.5 h for A. flavus, and 13 to 15.6 h for S. prolificans while awaiting formal MIC determination by the NCCLS reference method.     

Infections caused by Scedosporium spp

Scedosporium spp. are increasingly recognized as causes of resistant life-threatening infections in immunocompromised patients. Scedosporium spp. also cause a wide spectrum of conditions, including mycetoma, saprobic involvement and colonization of the airways, sinopulmonary infections, extrapulmonary localized infections, and disseminated infections. Invasive scedosporium infections are also associated with central nervous infection following near-drowning accidents. The most common sites of infection are the lungs, sinuses, bones, joints, eyes, and brain. Scedosporium apiospermum and Scedosporium prolificans are the two principal medically important species of this genus. Pseudallescheria boydii, the teleomorph of S. apiospermum, is recognized by the presence of cleistothecia. Recent advances in molecular taxonomy have advanced the understanding of the genus Scedosporium and have demonstrated a wider range of species than heretofore recognized. Studies of the pathogenesis of and immune response to Scedosporium spp. underscore the importance of innate host defenses in protection against these organisms. Microbiological diagnosis of Scedosporium spp. currently depends upon culture and morphological characterization. Molecular tools for clinical microbiological detection of Scedosporium spp. are currently investigational. Infections caused by S. apiospermum and P. boydii in patients and animals may respond to antifungal triazoles. By comparison, infections caused by S. prolificans seldom respond to medical therapy alone. Surgery and reversal of immunosuppression may be the only effective therapeutic options for infections caused by S. prolificans.     

Comparison of E-test and broth microdilution methods for antifungal drug susceptibility testing of molds

We compared the E test with a broth microdilution method, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 90 isolates of pathogenic molds (10 Absidia corymbifera, 10 Aspergillus flavus, 10 Aspergillus fumigatus, 10 Aspergillus niger, 10 Aspergillus terreus, 10 Exophiala dermatitidis, 10 Fusarium solani, 10 Scedosporium apiospermum, 5 Scedosporium prolificans, and 5 Scopulariopsis brevicaulis). Overall, there was 71% agreement between the results of the two methods for amphotericin B (E-test MICs within +/-2 log2 dilutions of broth microdilution MICs) and 88% agreement with the results for itraconazole. The overall levels of agreement (within +/-2 log2 dilutions) were >/=80% for 5 of the 10 species tested against amphotericin B and 8 of the 10 species tested against itraconazole. The best agreement between the results was seen with A. fumigatus and A. terreus (100% of results for both agents within +/-2 log2 dilutions). The poorest agreement was seen with S. apiospermum, S. prolificans, and S. brevicaulis tested against amphotericin B (20% of results within +/-2 log2 dilutions). In every instance, this low level of agreement was due to isolates for which the broth microdilution MICs were low but for which the E-test MICs were much higher. The E test appears to be a suitable alternative procedure for testing the susceptibility of Aspergillus spp. and some other molds to amphotericin B or itraconazole.     

The significance of blood cultures positive for emerging saprophytic moulds in cancer patients

The significance of blood cultures positive for emerging saprophytic moulds (e.g., Scedosporium apiospermum, Scedosporium prolificans, Paecilomyces spp.) was evaluated in 30 cancer patients (1996-2002). Diagnostic criteria proposed previously for evaluation of aspergillaemia were used. Blood cultures positive for emerging saprophytic moulds represented 1% of all positive fungal cultures. One case of catheter-related fungaemia was excluded. The remaining 29 cases consisted of true (n = 5), probable (n = 1), indeterminate (n = 7) fungaemia, and contamination (n = 16). True fungaemia was seen only in leukaemia patients and allogeneic bone marrow transplant recipients. S. apiospermum and S. prolificans were the commonest causes of true fungaemia.     

Isolation and serotyping of Streptococcus mutans from teeth and feces of children.

Streptococcus mutans were detected in the feces from 10 of 29 caries-active patients, aged 4 to 9 years. The percentage of S. mutans to the total counts of facultatively anaerobic streptococci on mitis salivarius agar (Difco Laboratories) varied from 0 to 72.5%. S. mutans were then isolated from dental plaque of sound teeth and carious dentin of the 10 subjects known to harbor S. mutans in the feces. The frequency distribution of various serotypes of these dental and fecal isolates of S. mutans was compared by the immunodiffusion technique. Of the total 1,047 isolates (290 isolates from feces, 289 from dental plaque, and 468 from carious dentin), type c isolates were most prevalent (ca. 66%). Serotype d, e, f, and g isolates were also found but in far lower frequencies. Plural serotypes of S. mutans were occasionally found in dental and fecal samples of a single subject. For two subjects, relatively rare serotypes of S. mutans in the population examined, serotype e, f, or g, were predominantly found in their fecal and dental samples and those of their siblings and mother, suggesting an intrafamilial transmission of S. mutans.     

Plasmid pattern analysis of Staphylococcal epidermidis isolates from patients with prosthetic valve endocarditis.

The electrophoretic pattern formed by individual bacterial plasmid DNA molecules of differing molecular size was evaluated as an epidemiological marker among isolates of Staphylococcus epidermidis from patients with prosthetic valve endocarditis (PVE). Purified covalently closed circular plasmid DNA was obtained from selected isolates, and 79% of the plasmids were found to be less than 10 megadaltons in size; only these small plasmids were sought in subsequent screening gels. Crude cell lysates obtained by a rapid lysis technique and screened by agarose gel electrophoresis revealed the presence of one or more small plasmids in 54 of 58 (93%) PVE isolates; 79% contained two or more. Among 45 plasmid-containing isolates from cases of sporadic PVE at three institutions there were no identical plasmid patterns, although several isolates differed by a single plasmid. In contrast, among nine isolates from a cluster of cases of PVE in Canada, two groups of three isolates each had identical plasmid patterns. Additional clinical data suggested that these isolates were epidemiologically related. Phage typing distinguished one of the groups with plasmid pattern identity, but not the other, from the three isolates with dissimilar patterns. Plasmid pattern analysis shows promise as an epidemiological marker for clinically important isolates of S. epidermidis.     

Breakthrough lung Scedosporium prolificans infection with multiple cavity lesions in a patient receiving voriconazole for probable invasive aspergillosis associated with monoclonal gammopathy of undetermined significance (MGUS)

Breakthrough non- Aspergillus mold infections among patients receiving the anti-mold azole antifungal agents like voriconazole or posaconazole have been increasingly reported. We report a case of lung Scedosporium prolificans infection with multiple cavities in a 58-year-old man with monoclonal gammopathy of undetermined significance (MGUS) during voriconazole treatment for probable invasive aspergillosis. Cultures of repeated sputum specimens yielded the same fungus until his death 83 days after diagnosis. S. prolificans should be considered in patients with breakthrough infections receiving voriconazole.     

Distribution of Staphylococcus sciuri subspecies among human clinical specimens, and profile of antibiotic resistance

The distribution of three subspecies comprising Staphylococcus sciuri was determined for a collection of 30 clinical isolates originating from Morocco, the United Kingdom, and France. The sources of these isolates were principally wounds, skin, and soft tissue infections. At the species level, the isolates were identified according to biochemical characteristics and at the subspecies level by the ribotyping technique. PCR analysis performed with the 16S-23S ribosomal DNA intergenic spacer was less powerful for subspecies differentiation. S. sciuri subsp. sciuri was the most frequent subspecies (21 isolates) found in the collection, whereas S. sciuri subsp. rodentium (seven isolates) and S. sciuri subsp. carnaticus (two isolates) were less common. mecA or a mecA-related gene was detected by PCR and Southern blot in all 30 S. sciuri isolates, supporting the suggestion that S. sciuri species are the natural reservoir of the mecA gene. While the linA/linA' gene coding for lincomycin resistance was present in five isolates, an uncharacterized gene for this resistance was suspected in seventeen other isolates.     

Studies on clinical isolates of coagulase-negative staphylococci resistant to methicillin. Evidence of cross-resistance between methicillin and cephalothin

The in vitro susceptibility to cephalothin and cefuroxime of 195 isolates of methicillin-resistant coagulase-negative staphylococci was determined by the agar-diffusion test, using 7.5% NaCl-supplemented agar. The distribution of the inhibition zone diameters for isolates of S. epidermidis (S. biotype 1) as well as for S. haemolyticus (S. biotype 4) was trimodal. While 4% of the isolates were found susceptible to cefuroxime, 39% of the S. epidermidis/S. hominis (S. biotype 1) isolates and 34% of the S. haemolyticus (S. biotype 4) isolates were found susceptible to cephalothin by this method. Eight of these isolates (six S. epidermidis, two S. haemolyticus) were selected for susceptibility testing by the tube-dilution method, together with four isolates (three S. haemolyticus, one S. epidermidis) found resistant to cephalothin by the agar-diffusion test. The first-mentioned isolates were all found susceptible to cephalothin with MICs less than or equal to 2 micrograms/l, while the last-named all were resistant with MICs greater than or equal to 16 micrograms/ml. Population analyses revealed sub-populations of highly resistant bacteria in all methicillin-resistant isolates of S. epidermidis (S. biotype 1), as well as in all isolates of S. haemolyticus (S. biotype 4). We thus concluded that methicillin-resistance in isolates of coagulase-negative staphylococci implies resistance to cephalosporins and that the difference between S. epidermidis and S. haemolyticus as regards cephalosporin-susceptibility is quantitative and not qualitative. Eighty-nine per cent of the 195 methicillin-resistant isolates in this study were resistant to penicillin and at least one more antibiotic. We therefore think that resistance to penicillin and one or more non-beta-lactam antibiotics strongly suggests methicillin-resistance and that such isolates should be further tested on hypertonic media.     

Use of PCR to study epidemiology of Serratia marcescens isolates in nosocomial infection.

A method to characterize strains of Serratia marcescens based on the PCR amplification of enterobacterial repetitive intergenic consensus sequences has been developed. The PCR fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during an outbreak of pneumonia from seven mechanically ventilated patients, and its result indicated that the outbreak was due to the spread of two epidemic strains. This technique was validated by comparison with rRNA gene restriction analysis. There was complete concordance between these two techniques in discriminating the outbreak-related strains from epidemiologically unrelated isolates. Typing with both biochemical profile and antibiogram profile, though simple, was found to be less reliable than genotyping. The results show that this enterobacterial repetitive intergenic consensus PCR provides a rapid and simple means of typing S. marcescens isolates for epidemiologic studies.     

Epidemiological Investigation of Vaginal Saccharomyces cerevisiae Isolates by a Genotypic Method

Saccharomyces cerevisiae is a ubiquitous, ascomycetous yeast, and vaginitis caused by this organism has been reported only very rarely. The aim of the present investigation was to assess the epidemiological relatedness of a group of vaginal and commercial S. cerevisiae isolates by a previously reported genetic typing method, which divided the isolates into two broad groups with numerous subtypes. Nineteen S. cerevisiae isolates obtained from patients suffering from vaginitis and four isolates from commercial products in the same city were analyzed. The cellular DNA from each isolate was digested with the restriction endonuclease EcoRI, and restriction fragment length polymorphisms were generated by horizontal gel electrophoresis. The results showed that although vaginal isolates did not cluster in any particular genetic subtype, multiple patients were infected with indistinguishable strains (there were nine distinct strains among 23 isolates). For two of three patients, all three with two episodes of S. cerevisiae vaginitis, different strains were isolated during the recurrence of this disease. Three other patients with indistinguishable isolates were epidemiologically related in that two were practitioners in the same clinic and the third was a patient at this clinic. We also found that one commercial strain was indistinguishable from the strain isolated from three different women at the time that they were suffering from vaginitis. The findings of the present study suggest that some S. cerevisiae strains may possess properties permitting persistence in the human host. Furthermore, person-to-person contact and the proliferation of the use of S. cerevisiae as a health-food product, in home baking, and in home brewing may be a contributing factor in human colonization and infection with this organism.     

Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients

In total, 40 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients were included in this study. Twenty of these were collected in 1994 and 1997, from six CF patients, and the rest were collected from different CF patients in 2000 and 2001. The relative expression of mRNA for the efflux pump protein MexY was determined by real-time PCR and correlated with susceptibilities to amikacin and tobramycin. The chromosomal genes mexZ , rplY , galU , PA5471 and nuoG , which were found to have a role in the gradual increase in MICs of aminoglycoside antibiotics in laboratory mutants of P. aeruginosa , were analysed. MexY mRNA overproduction was found in 17/20 isolates collected in 1994 and 1997, and was correlated with decreased susceptibility to aminoglycosides. Alteration of the MexXY–OprM efflux system has been the main mechanism of resistance to aminoglycoside antibiotics in CF P. aeruginosa isolates over the 3-year period. In several isolates, expression of the PA5471 gene product might have some effect on elevated MICs of aminoglycosides. Inactivation of rplY , galU and/or nuoG may explain the gradual increase in MICs of aminoglycosides in laboratory mutants but probably not in the CF environment, as rplY and galU were unaltered in all isolates, and nuoG was not expressed in only one isolate. No 16S rRNA A-site mutations were found in any of the four copies of the gene in 13 investigated isolates.