Validation of a New Web Application for Identification of Fungi by Use of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has emerged as a reliable technique to identify molds involved in human diseases, including dermatophytes, provided that exhaustive reference databases are available. This study assessed an online identification application based on original algorithms and an extensive in-house reference database comprising 11,851 spectra (938 fungal species and 246 fungal genera). Validation criteria were established using an initial panel of 422 molds, including dermatophytes, previously identified via DNA sequencing (126 species). The application was further assessed using a separate panel of 501 cultured clinical isolates (88 mold taxa including dermatophytes) derived from five hospital laboratories. A total of 438 (87.35%) isolates were correctly identified at the species level, while 26 (5.22%) were assigned to the correct genus but the wrong species and 37 (7.43%) were not identified, since the defined threshold of 20 was not reached. The use of the Bruker Daltonics database included in the MALDI Biotyper software resulted in a much higher rate of unidentified isolates (39.76 and 74.30% using the score thresholds 1.7 and 2.0, respectively). Moreover, the identification delay of the online application remained compatible with real-time online queries (0.15 s per spectrum), and the application was faster than identifications using the MALDI Biotyper software. This is the first study to assess an online identification system based on MALDI-TOF spectrum analysis. We have successfully applied this approach to identify molds, including dermatophytes, for which diversity is insufficiently represented in commercial databases. This free-access application is available to medical mycologists to improve fungal identification.

Validation of a modified algorithm for the identification of yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

Optimising antifungal treatment requires the fast and species-specific identification of yeast isolates. We evaluated a modified protocol for the rapid identification of clinical yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) technology. First, we evaluated a simplified extraction procedure using 54 clinical yeast isolates. Second, we validated a new protocol with this simplified extraction procedure and lower identification threshold by analysing 167 isolates with either MALDI-TOF or conventional identification techniques. MALDI-TOF analysis with both the standard and short extraction procedure yielded identical identification results, although the log-scores were lower with the latter. With the modified protocol, 163/167 (97.6%) isolates showed a correct identification as compared to conventional identification techniques. A total of 135 out of the 163 (82.8%) correct identifications showed log-scores above 1.7, which we considered as the minimum log-score for secure species identification. The rapid identification of clinical yeast isolates is crucial in patient management. The MALDI-TOF technique using a short extraction procedure can be an alternative for the labourious standard procedure, although the log-scores will be lower. The identification of clinical yeast isolates with the modified protocol is a practical and accurate alternative for conventional identification techniques. If the isolate shows a log-score below 1.7, the standard extraction procedure should be used.

Breakthrough Saprochaete capitata infections in patients receiving echinocandins: case report and review of the literature.

We report a rare case of a fatal Saprochaete capitata breakthrough infection in a patient with acute myeloid leukemia receiving empirical caspofungin therapy. S. capitata is an uncommon, yet emerging cause of invasive infections, especially in patients with haematological malignancies. Blood cultures from our patient yielded S. capitata which was found to be resistant, in vitro, to caspofungin. We consecutively reviewed all published cases of breakthrough infections caused by S. capitata in patients receiving echinocandins. S. capitata should be considered in those patients who remain febrile or who develop invasive mould infections while under echinocandin therapy.

10th survey of antimicrobial resistance in noninvasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2007-2008.

OBJECTIVES: The aim of the study was to evaluate the antibiotic resistance in noninvasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2008-2007. METHOD: Four hundred and forty eight unduplicated isolates collected by 15 laboratories were tested by microdilution following CLSI. RESULTS: Insusceptibility rates (I+R) were as follows: penicillin G (PEN) 11.6% (4.0% R), ampicillin 11.4% (4.0% R), amoxicillin+/-clavulanic acid 0, cefaclor 10.3% (9.6% R), cefuroxime 9.2% (8.7% R), cefuroxime-axetil 8.7% (7.8% R), cefotaxime, ceftazidime and cefepime 2.0% (0% R), imipenem 2.5% (0% R), ciprofloxacin and ofloxacin 5.1% (0.4% R), levofloxacin 0.7% (0.4% R), moxifloxacin 0.4% (0.2% R), erythromycin (ERY) 29.7% (29.2% R), azithromycin 29.7% (28.8% R), telithromycin 0%, clindamycin 26.3% (25.4% R) and tetracycline (TET) 21.9% (16.5% R). From 2001 to 2008, a significant decrease in penicillin-insusceptibility (21.0% to 11.6%), penicillin-resistance (9.7% to 4.0%) and ciprofloxacin-insusceptibility (11.2% to 5.1%) was found. Cross-resistance between penicillin and other betalactams in penicillin-insusceptible isolates was incomplete: all these isolates remained fully susceptible to amoxicillin. Erythromycin-insusceptibility was significantly higher in children than in adults (43.9%/27.4%), while penicillin-insusceptibility significantly higher in Brussels than in the Flanders (22.9%/8.1%). The commonest resistance phenotype was ERY-TET (12.7%) followed by ERY (7.4%) and PEN-ERY-TET (5.8%). Capsular types 19 (25%), 14 (19.3%), 23 (15.4%) and 15 (13.5%) were the most important in penicillin-insusceptible. CONCLUSION: We noted a decrease in resistance to the majority of the compounds. Insusceptibility rates were higher in children than in adults and the difference between the north and the south of Belgium became less marked.

Histoplasmosis in Europe: report on an epidemiological survey from the European Confederation of Medical Mycology Working Group.

The purpose of this survey was to systematically collect data on individuals with histoplasmosis in Europe over a 5-year period (from January 1995 to December 1999). This included information on where and how the infection was acquired, the patient's risk factors, the causative organism, how the infection was diagnosed and what therapy the patients received. Data were sent on a standardized survey form via a national convenor to the coordinator. During the survey, 118 cases were reported, with 62 patients having disseminated disease, 31 acute pulmonary infection, chronic pulmonary infection in 6 and localized disease in 2 patients. For 17 patients, the diagnosis of histoplasmosis was incidental, usually secondary to investigations for lung cancer. Most patients had travelled to known endemic areas, but 8 patients (from Italy, Germany and Turkey) indicated that they had not been outside their countries of origin and hence these cases appear to be autochthonous. Notable observations during the survey were the reactivation of the disease up to 50 years after the initial infection in some patients and transmission of the infection by a transplanted liver. Itraconazole was the most commonly used therapy in both pulmonary and disseminated disease. The observation of autochthonous cases of disease suggests that the endemic area of histoplasmosis is wider than classically reported and supports continued surveillance of the disease throughout Europe.

Infection of the thyroid gland caused by Campylobacter fetus subsp. fetus.

We report what we consider to be the first case of an abscess of the thyroid gland due to Campylobacter fetus subsp. fetus (C. fetus) in a patient suffering from hyperthyroidism. C. fetus is known as a rare and opportunistic pathogen in humans, causing a broad variety of systemic infections. Acquisition by humans is thought to occur through contact with animals or animal products and to start as a gastro-intestinal colonization. The detection of C. fetus in stool is challenging, since culture efforts are generally directed in order to fulfil growth requirements of C.jejuni, a much more common enteric pathogen. Detection of C. fetus in non-stool samples is even more challenging since routine culture doesn't imply prolonged incubation (>72h), selective media and microaerophilic conditions. It is therefore not unlikely that human infections caused by C. fetus occur more often than generally assumed.

Surveillance of antibiotic resistance in non invasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winters 2003 and 2004.

A total of 391 and 424 non-invasive isolates of Streptococcus pneumoniae collected by 15 laboratories during the 2003 and 2004 survey were tested for their susceptibility by a microdilution technique following NCCLS recommendations. Insusceptibility rates (IR) in the two surveys (2003/2004) were as follows: penicillin 15.0/14.7% [8.4/6.4% Resistance (R)], ampicillin 17.4/14.6% (R 9.0/7.1%), amoxicillin +/- clavulanic acid 2.6/1.2 % (R 0/0%), cefaclor 14.3/14.1% (R 11.5/13.4%), cefuroxime 13.6/12.7% (R 10.5/11.8%), cefuroxime-axetil 10.5/11.8% (R 10.0/9.2%) (breakpoints based on 250 mg), cefotaxime 4.9/6.2% (R 1.3/2.4%), ceftazidime NotTested (NT)/6.4 (R NT/2.6%), cefepime NT/6.4 (R NT/2.6%), imipenem 7.7/8.9 % (R 1.8/1.4%), ertapenem 0.8/NT% (R O/NT%), ciprofloxacin 13.8/9.0% (R 4.3/2.4%), levofloxacin 3.3/2.8% (R 1.5/0.2%), moxifloxacin 0.6/0.2% (R 0.3/0%), ofloxacin 13.5/9.0% (R 4.3/2.4%), erythromycin 26.1/24.7% (R 25.3/24.5%), azithromycin 25.4/24.7% (R 24.6/24.5%), telithromycin 0.8/0.2% (R 0.5/0%), clindamycin 21.2/18.4% (R 19.2/17.7%) and tetracycline 32.3/22.1% (R 29.2/19.3%). There were only minor differences in resistance rates according to age, sample site, admission type (i.e. ambulatory, hospitalized or long-term care facility patients), gender and geographic origin. Overall, telithromycin (MIC50, MIC90 in 2003/2004: 0.015 microg/ml, 0.12 microg/ml/ 0.008,0.06 respectively), ertapenem (0.03; 0.25/NT), moxifloxacin (0.06; 0.25/0.06, 0.12), and amoxicillin +/- clavulanic acid (0.03; 0.25/0.015, 0.5) were the most active compounds in both surveys. In 2003, the most common resistance phenotype was isolated insusceptibility to tetracycline (10.5%) followed by combined insusceptibility to erythromycin and tetracycline (9.3%). Erythromycin-tetracycline resistance (10.4%) was the most common in 2004. Isolates showing resistance to an antibiotic were significantly more present in 2003 than in 2004 (50.4% versus 40.8%). In penicillin-insusceptible isolates, MICs of all beta-lactams were increased but cross-resistance between penicillin and other beta-lactams in the penicillin-insusceptible isolates was not complete. In the 2003 survey, most of these isolates remained fully susceptible to ertapenem (94.9%) and amoxicillin +/- clavulanic acid (83.1%). In the 2004 survey, 91.9% of the penicillin insusceptible isolates remained susceptible to amoxicillin +/- clavulanic acid. In both surveys, the most common serotypes in penicillin insusceptible isolates were 14, 23,19 and 9 (20.0%, 20.0%, 16.4% and 10.9% respectively in 2003; 41.6%, 11.7%, 15.0% and 18.3% respectively in 2004).

Three cases of destructive native valve endocarditis caused by Staphylococcus lugdunensis.

Described here are three cases of acute native valve endocarditis due to the coagulase-negative pathogen Staphylococcus lugdunensis with serious complications. Two of the three patients died despite optimal antibiotic therapy and cardiovascular surgery. These cases demonstrate the aggressive nature of S. lugdunensis and emphasize the importance of identifying coagulase-negative staphylococci to the species level and not considering the isolation of S. lugdunensis from normally sterile body fluids as contamination. On the contrary, when this organism is found in patients with endocarditis, early surgery should be considered. The possibility that this organism could be misidentified as S. aureus because of "autocoagulation" and that commercial identification systems may misidentify it as S. haemolyticus, S. hominis or S. warneri should also be remembered.

Surveillance of antibiotic resistance in clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2000-2001.

A total of 314 isolates of Streptococcus pneumoniae collected by 10 different laboratories were tested for their susceptibility by using a microdilution technique following NCCLS recommendations. The following antibiotics were included: penicillin, ampicillin, amoxicillin, amoxicillin/clavulanate, cefaclor, cefuroxime, cefotaxime, imipenem, ciprofloxacin, gemifloxacin, levofloxacin, erythromycin, clarithromycin, azithromycin, miocamycin, clindamycin and tetracycline. The insusceptibility rate (IR) to penicillin was 21.0% [10.8% intermediate (> or = 0.12-1 microgram/mL) and 10.2% high-level (> or = 2 micrograms/mL)], to cefotaxime 7.3% [3.5% intermediate (> or = 1 microgram/mL) and 3.8% high-level (> or = 2 micrograms/mL)], to imipenem 3.8% [3.8% intermediate (> or = 0.25-0.5 microgram/mL) and 0% high-level (> or = 1 microgram/mL)], to ciprofloxacin 11.2% [8.3% intermediate (2 micrograms/mL) and 3.9% high-level (> or = 4 micrograms/mL)], to erythromycin 30.3% [3.5% intermediate (0.5 microgram/mL) and 26.8% high-level (> or = 1 microgram/mL)] and to tetracycline 38.5% [0.9% intermediate (4 micrograms/mL) and 37.6% high-level (> or = 8 micrograms/mL)]. No decreased susceptibility was found for gemifloxacin (> or = 0.5 microgram/mL). This compound was the most active with MIC50, MIC90 and an IR of 0.015 microgram/mL, 0.03 microgram/mL and 0% respectively, followed by amoxicillin/clavulanate, amoxicillin and imipenem (MIC50, MIC90 and IR: 0.015 microgram/mL, 1 microgram/mL, 1.6%/0.015 microgram/mL, 1 microgram/mL, 1.9%/0.008 microgram/mL, 0.12 microgram/mL, 3.8% respectively). Compared to the 1999 surveillance, penicillin and tetracycline-insusceptibility increased with 4.9% and 15.6% respectively, while cefotaxime, erythromycin and ciprofloxacin insusceptibility decreased with 5.4%, 5.8% and 4.4% respectively. MICs of all beta-lactams rose with those of penicillin for penicillin-insusceptible isolates. Imipenem, cefotaxime, amoxicillin and amoxicillin/clavulanate were generally 4, 2, 1 and 1 doubling dilutions respectively more potent than penicillin on these isolates while ampicillin, cefuroxime and cefactor were generally 1, 2 and 4 dilutions respectively [table: see text] less potent. Most penicillin-insusceptible isolates remained fully susceptible to amoxicillin/clavulanate (92.4%), amoxicillin (90.9%) and imipenem (81.8%). Erythromycin-tetracycline insusceptibility was the most common resistance phenotype (14.3%). Three- and four-fold resistance was found in 12.4% and 1.6% respectively of the isolates. Most penicillin-insusceptible isolates were of capsular types 14 (22.7%), 23 (21.2%), 6 (18.2%), 9 (13.6%) and 19 (12.1%).

Subacute endocarditis due to Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae is a rare cause of endocarditis. Most cases were observed in people working with animals. We report a case in a 45-year old man without any exposure to animals. He was admitted to our hospital because of dyspnoea. Blood cultures were drawn following fever on day 8 of hospitalisation. Erysipelothrix rhusiopathiae was cultured and echocardiography showed a vegetation on the mitral valve. Appropriate antibiotic therapy and surgical treatment led to a good outcome of the infection.

A pulmonary infection caused by Coprinus cinereus (Hormographiella aspergillata) diagnosed after a neutropenic episode.

In a patient treated for a non-Hodgkin's lymphoma, a lung abscess caused by Hormographiella aspergillata (stat. anamorph. Coprinus cinereus) was diagnosed using an ultrasound-guided puncture of the lesion. The patient appeared to respond to amphotericin B, but at the same time was also recovering from her neutropenic episode. The extent to which each of these two factors explains the complete resolution of the infection is unclear. Expert classical morphological examination and molecular typing methods were needed to identify this filamentous basidiomycetous fungus.

Comparative in vitro activity of temocillin and other antimicrobial agents against Enterobacteriaceae isolated from patients admitted to five Belgian hospitals.

Temocillin, a methoxy-derivative of the broad-spectrum penicillin, ticarcillin, has been introduced into clinical practice in Belgium in 1988. Since then, not many surveys of its in vitro activity have been published. This study addresses this issue in a prospective collection of 300 consecutive Gram-negative isolates originating from in-patients in five general hospitals throughout Belgium. In addition to temocillin, seven common antibiotics were tested: amoxicillin-clavulanate, piperacillin-tazobactam, cefotaxime, aztreonam, meropenem, ciprofloxacin and amikacin. Meropenem appeared to exhibit the best activity overall, whereas amoxicillin-clavulanate scored the worst. Cumulative MIC plot for two subsets of organisms are given: temocillin, meropenem and cefotaxime are the most active on E. coli and Klebsiella spp., while a significant percentage is resistant to ciprofloxacin and amoxicillin-clavulanate. In the group of inducible Enterobacteriaceae, temocillin, meropenem and amikacin are the most active drugs, while the activity of amoxicillin-clavulanate, piperacillin-tazobactam, cefotaxime and ciprofloxacin is largely decreased. Taking this well preserved in vitro activity of temocillin into account, and looking at its convenient pharmacokinetics and low cost of acquisition, this drug may prove a useful alternative in the treatment of severe nosocomial infections.

Disseminated infection by Scedosporium prolificans: an emerging fatality among haematology patients. Case report and review.

We describe a case of proven disseminated infection by Scedosporium prolificans in a profoundly neutropenic patient. The patient presented with a fever unresponsive to broad-spectrum antibiotics, endophthalmitis, respiratory failure and a renal abscess. The organism was isolated from bronchoalveolar lavage fluid and from pus obtained through a sterile puncture. Review of the English-language literature identified 28 additional cases; these occurred exclusively in severely neutropenic patients (predominantly leukaemia) and in transplant recipients. Apart from two or possibly three cases, dissemination was uniformly fatal due to persistent neutropenia and inherited resistance of these pathogens to currently available antifungal drugs. At present, the optimal treatment of S. prolificans infections is unknown, but reversal of the underlying deficient immune status appears of great importance.

In vitro study on the antimicrobial activity of various antibiotics against clinical isolates of Streptococcus pneumoniae from Belgium collected during winter 1998-1999.

A total of 205 isolates of Streptococcus pneumoniae obtained from 10 different centres were included in this study. The susceptibilities to penicillin, ampicillin, amoxicillin, amoxicillin/clavulanic acid, cefaclor, cefuroxime, cefotaxime, imipenem, ciprofloxacin, gemifloxacin, grepafloxacin, levofloxacin, trovafloxacin, erythromycin, clarithromycin, miocamycin, clindamycin and tetracycline were determined by a microdilution technique following NCCLS recommendations. Decreased susceptibility to penicillin was 16.1% [6.8% intermediate (0.12-1 microgram/mL) and 9.3% high-level (> or = 2 micrograms/mL)], cefotaxime insusceptibility (> or = 1 microgram/mL) 12.7%, ciprofloxacine insusceptibility (> or = 2 micrograms/mL) 15.6% with 1.5% of high level resistance (> or = 4 micrograms/mL), erythromycin insusceptibility (> or = 0.5 microgram/mL) 36.1% and tetracycline insusceptibility (> or = 4 micrograms/mL) 22.9%. Decreased susceptibility to cefotaxime was found in 78.8% of the penicillin-insusceptible isolates. No decreased susceptibility was found for gemifloxacin (> or = 0.5 microgram/mL) and trovafloxacin (> or = 1 microgram/mL). Compared to the 1996-1997 surveillance, penicillin, cefotaxime and erythromycin insusceptibility rose by 3.8%, 5.2% and 5.0% respectively, while tetracycline insusceptibility decreased with 8.2%. MICs of all beta-lactams rose with those of penicillin for penicillin-insusceptible isolates. Amoxicillin +/- clavulanate, cefotaxime and imipenem were generally 1, 1 and 5 doubling dilutions respectively more potent than penicillin on these isolates. Penicillin, ampicillin and cefuroxime were equally active while cefaclor was generally 5 dilutions less potent. Most penicillin-insusceptible isolates remained fully susceptible to amoxicillin +/- clavulanate and imipenem. The penicillin-insusceptible isolates were 36.4%, 27.3% and 3.0% co-insusceptible to erythromycin, erythromycin plus tetracycline and tetracycline respectively. A subpopulation of 52 isolates obtained from children aged < or = 3 years was also studied. Compared to the other isolates we found a statistically significant increase in insusceptibility for penicillin, cefaclor, cefuroxime, erythromycin, clarithromycin and tetracycline while a significant decrease was found for ciprofloxacin.

The hypereosinophilic syndrome after residence in a tropical country: report of 4 cases.

Severe eosinophilia may be complicated by acute or chronic visceral damage. The underlying origin of the hypereosinophilia may be infectious, allergic, toxic, malignant or systemic (the secondary or reactive hypereosinophilic syndrome), but in a number of cases no cause can be found (the idiopathic hypereosinophilic syndrome). We describe 4 cases with hypereosinophilia and secondary visceral damage after residence in a tropical region. In three cases a helminthic infection was the obvious cause, the brain and the heart were the target organs. After treatment of the infection both the hypereosinophilia and the neurological and cardiac lesions disappeared. The fourth patient died of multi-organ disease. No definite trigger of the hypereosinophilia could be found. We discuss clinical findings, necessary investigations and therapeutic strategies.