Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and accurate identification of Pseudallescheria/Scedosporium species.

An increasing number of infections due to Pseudallescheria/Scedosporium species has been reported during the past decades, both in immunocompromised and immunocompetent patients. Additionally, these fungi are now recognized worldwide as common agents of fungal colonization of the airways in cystic fibrosis patients, which represents a risk factor for disseminated infections after lung transplantation. Currently six species are described within the Pseudallescheria/Scedosporium genus, including Scedosporium prolificans and species of the Pseudallescheria/Scedosporium apiospermum complex (i.e. S. apiospermum sensu stricto, Pseudallescheria boydii, Scedosporium aurantiacum, Pseudallescheria minutispora and Scedosporium dehoogii). Precise identification of clinical isolates at the species level is required because these species differ in their antifungal drug susceptibility patterns. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF)/mass spectrometry (MS) is a powerful tool to rapidly identify moulds at the species level. We investigated the potential of this technology to discriminate Pseudallescheria/Scedosporium species. Forty-seven reference strains were used to build a reference database library. Profiles from 3-, 5- and 7-day-old cultures of each reference strain were analysed to identify species-specific discriminating profiles. The database was tested for accuracy using a set of 64 clinical or environmental isolates previously identified by multilocus sequencing. All isolates were unequivocally identified at the species level by MALDI-TOF/MS. Our results, obtained using a simple protocol, without prior protein extraction or standardization of the culture, demonstrate that MALDI-TOF/MS is a powerful tool for rapid identification of Pseudallescheria/Scedosporium species that cannot be currently identified by morphological examination in the clinical setting.

Prospective survey of acute osteoarticular infections in a French paediatric orthopedic surgery unit.

The epidemiology of acute paediatric osteoarticular infections (OAI) has recently evolved, mainly due to the improvement of microbiological diagnosis. We conducted a prospective study to analyse the recent epidemiology and the clinical evolution of paediatric OAI in order to validate the adequacy of our probabilistic first-line antibiotic treatment (intraveinous cefamandole + gentamicin). All children suspected of community acquired OAI were included and followed-up for 3 years. The etiologic diagnosis was based on blood cultures, joint aspirations and bone punctures. All osteoarticular (OA) samples were systematically inoculated into blood culture bottles. Real-time universal 16S rRNA and PCR targeted on Staphylococcus aureus, Kingella kingae, Streptococcus pneumoniae and Streptococcus pyogenes were performed twice a week. From 17 March 2007 to 26 February 2009, 98 septic arthritis, 70 osteomyelitis, 23 osteoarthritis and six spondylodiscitis were analysed. A portal of entry was suspected in 44% of cases, including 55% of otorhinolaryngological infections. C reactive protein was the most sensitive inflammatory marker. PCR increased by 54% the performance of bacteriological diagnosis. Among the patients completely investigated (blood culture and OAI samples), there were 63% documented OAI. The main pathogens found were K. kingae (52%), S. aureus (28%), S. pyogenes (7%), S. pneumoniae (3%) and Streptococcus agalactiae (2%). All isolated bacteria were sensitive to the probabilist treatment and outcome was favorable. PCR has significantly improved the performance and the delay of IOA diagnosis in children, for which K. kingae turned out to be the first causative agent. The probabilistic treatment was active against the main bacteria responsible for paediatric OAI.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species.

New Aspergillus species have recently been described with the use of multilocus sequencing in refractory cases of invasive aspergillosis. The classical phenotypic identification methods routinely used in clinical laboratories failed to identify them adequately. Some of these Aspergillus species have specific patterns of susceptibility to antifungal agents, and misidentification may lead to inappropriate therapy. We developed a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based strategy to adequately identify Aspergillus species to the species level. A database including the reference spectra of 28 clinically relevant species from seven Aspergillus sections (five common and 23 unusual species) was engineered. The profiles of young and mature colonies were analysed for each reference strain, and species-specific spectral fingerprints were identified. The performance of the database was then tested on 124 clinical and 16 environmental isolates previously characterized by partial sequencing of the ß-tubulin and calmodulin genes. One hundred and thirty-eight isolates of 140 (98.6%) were correctly identified. Two atypical isolates could not be identified, but no isolate was misidentified (specificity: 100%). The database, including species-specific spectral fingerprints of young and mature colonies of the reference strains, allowed identification regardless of the maturity of the clinical isolate. These results indicate that MALDI-TOF MS is a powerful tool for rapid and accurate identification of both common and unusual species of Aspergillus. It can give better results than morphological identification in clinical laboratories.

[Bordetella bronchiseptica-associated acute chest syndrome in a child with sickle cell disease]. / Syndrome thoracique aigu associé à une infection àBordetella bronchiseptica chez un enfant drépanocytaire.

We describe a case of acute chest syndrome associated with Bordetella bronchiseptica pneumonia in a child with sickle cell disease. B. bronchiseptica is recognized as an important pathogen of the respiratory tract for a large variety of animal species. This zoonotic agent has been frequently associated with chronic and recurrent infections. In humans, the bacterium acts as an opportunistic pathogen affecting mostly immunocompromised patients or those with preexisting respiratory diseases. This case and literature review provides an opportunity to discuss the risk factors, treatment, follow-up, and prevention of such zoonotic infections in the context of a lack of cross-protection of new pertussis vaccines.

Detection of circulating Aspergillus fumigatus DNA by real-time PCR assay of large serum volumes improves early diagnosis of invasive aspergillosis in high-risk adult patients under hematologic surveillance.

Detection of galactomannan antigen (GMA) in serum is the standard assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematological disorders. Detection of Aspergillus DNA in serum has been proposed, but its sensitivity is lower than that of GMA when small serum volumes (SSV) are used. In this study, we investigated whether extraction of DNA from large serum volumes (LSV) improves diagnostic yield. In a 13-month prospective study, we compared the performances of twice-weekly screening of serum for GMA by an enzyme immunoassay and weekly screening for Aspergillus fumigatus DNA by a real-time PCR (RT-PCR) assay of 1.0 ml (LSV) or 100 mul (SSV) of serum. We included 124 patients (138 treatment episodes), with 17 episodes of EORTC (European Organization for Research and Treatment of Cancer)/MSG (Mycoses Study Group)-documented IA. In all, 1,870 samples were screened for GMA. The sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV, respectively) of GMA for IA were 88.2%, 95.8%, 75%, and 98.3%, respectively. We screened 938 samples for Aspergillus DNA by using LSV; 404 of these samples were also tested with SSV. The Se, Sp, PPV, and NPV of RT-PCR were 100%, 96.7%, 81%, and 100%, respectively, with LSV and 76.5%, 96.7%, 81.3%, and 95.6%, respectively, with SSV. DNA detection gave a positive result when performed on LSV in two cases of IA where the GMA assay result remained negative. Furthermore, in four IA cases, DNA was detected earlier than GMA. The use of LSV for extraction improved the performance of the RT-PCR, which appears highly sensitive and specific for the early diagnosis of IA in high-risk patients with hematological disorders.

[Rapid antigen detection tests for diagnosis of group A streptococcal pharyngitis: comparative evaluation of sensitivity and practicability of 16 in vitro diagnostics medical devices performed in July 2002 by the French health products safety agency (Afssaps) as part of its market control mission]. / Tests de diagnostic rapide des angines à streptocoque du groupe A : étude comparative de la sensibilité et de la praticabilité de 16 dispositifs médicaux de diagnostic in vitro dans le cadre d'un contrôle du marché effectué par l'Afssaps en juillet 2002.

Within the scope of its health products control mission, the French Health Products Safety Agency (Afssaps) collaborating with two expert's sites, has assessed the 16 tests available on the French market in 2002 for rapid diagnosis of the Streptococcus A tonsillitis. The purpose of this study was to verify the reliability and rapidity of these tests and to give some information to the users about their analytical criteria and practicability characteristics. The analytical study has been performed on a same panel of four reference strains of Streptococcus pyogenes dilutions to determine the limit of detection of all the reagents in the same condition of methodology. The limit of detection has been calculated with the results expressed in colony forming unit by ml (CFU/ml). The practicability study has permitted to analyze the quality of the presentation, the easiness of the final reading and of performing tests. A score has been established for each rapid test. A classification of the analytical sensitivity (limit of detection) and practicability (score) of these 16 devices has been established. The limit of detection of the reagents giving the best results allows the detection of the lowest bacterial concentration of the panel which is 10(5) CFU/ml. Regarding practicability, the results suggest that, the immunochromatographic strip methods have the best score in a view with the use by a non medical laboratory.

Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target.

Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences. Sequence analysis revealed that the staphylococcal sodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.

Characterization of the Tn916-like transposon Tn3872 in a strain of abiotrophia defectiva (Streptococcus defectivus) causing sequential episodes of endocarditis in a child.

Clinical blood isolates from three sequential episodes of endocarditis occurring over a 6-month period in a child with a malformative cardiopathy were investigated. All isolates identified as Abiotrophia defectiva were resistant to erythromycin-clindamycin and to tetracycline-minocycline, due to the presence of sequences homologous to the erythromycin resistance gene ermB and to the tetracycline resistance gene tet(M), respectively. These resistance genes were located on a chromosomally borne composite Tn916-related transposon. These results demonstrate the involvement of conjugative transposons in the dissemination of antibiotic resistance in the genus Abiotrophia.

Outbreak of nosocomial urinary tract infections due to Pseudomonas aeruginosa in a paediatric surgical unit associated with tap-water contamination.

An outbreak of 14 cases of urinary tract infections by Pseudomonas aeruginosa, including six symptomatic infections, occurred from September to November 1994 in a paediatric surgical unit. During the outbreak, urine samples from patients and multiple samples from the environment of patients were tested for the presence of P. aeruginosa. Bacterial isolates were studied by pulsed-field gel electrophoresis. Genotypic analysis showed that most of the isolates from children were different. Multiple P. aeruginosa isolates were also found in the tap water, as the only putative source of contamination. Two of these isolates were identified in two infected patients, indicating possible direct contamination of patients via tap water and this was related to the distal colonization of faucets. Bacteria were eradicated from tap water by replacement of taps. The cluster of cases of P. aeruginosa urinary infection was, therefore, related to multiple contaminations through tap water. These results illustrate an unexpected risk of nosocomial infection and emphasizes the importance of checking tap water to prevent bacterial contamination through handwashing in contaminated water.

A novel extended-spectrum TEM-type beta-lactamase (TEM-52) associated with decreased susceptibility to moxalactam in Klebsiella pneumoniae.

Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum beta-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from blaTEM-1 by three mutations leading to the following amino acid substitutions: Glu104-->Lys, Met182-->Thr, and Gly238-->Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated blaTEM-52. The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (Ki, 0.16 microM) was 10-fold higher than that of TEM-3 (Ki, 1.9 microM). Allelic replacement analysis revealed that the combination of Lys104, Thr182, and Ser238 was responsible for the increase in the MICs of MOX for the TEM-52 producers.

Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase.

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

Wolman's disease: The King Faisal Specialist Hospital and Research Centre experience.

BACKGROUND: Wolman's disease is a rare autosomal recessive lysosomal storage disease. A recent review indicates that approximately 50 patients have been reported in the world. Reports of patients from the Arabian peninsula are rare due to lack of awareness among pediatricians. PATIENTS AND METHODS: We retrospectively reviewed the clinical, radiological, biochemical and histopathological findings of four Saudi patients diagnosed with Wolman's disease at King Faisal Specialist Hospital and Research Centre. The diagnosis was confirmed by deficient acid lipase activity in the leukocytes and fibroblasts, which was measured using 4-methylumbelliferyl palmitate. RESULTS: All patients were failing to thrive with progressive hepatosplenomegaly. Abdominal x-ray revealed calcifications which were confirmed on abdominal CT scan. Peripheral blood film showed vacuolated lymphocytes and the bone marrow aspiration showed foamy histiocytes. Liver biopsy in one patient showed marked steatosis and elliptical empty clefs predominantly in the Kupffer cells, indicating cholesterol storage in the reticulo-endothelial cells. The acid lipase activity was less than 6% in all patients. CONCLUSION: In all suspected cases of Wolman's disease, a plain abdominal x-ray should be obtained to check for the typical pattern of adrenal calcification characteristic of the disease, especially in any young infant with failure to thrive and progressive hepatosplenomegaly.

Idiopathic focal foveolar hyperplasia in infants.

Idiopathic focal foveolar hyperplasia (FFH) is a rare cause of gastric outlet obstruction in infants. We present two cases, including the first reported association with infantile hypertrophic pyloric stenosis. The diagnostic ultrasound appearance of FFH is described. Optimal treatment is surgical excision of the involved mucosa with pyloroplasty or pyloromyotomy.

Emergence of vancomycin resistance in the genus Streptococcus: characterization of a vanB transferable determinant in Streptococcus bovis.

Streptococcus bovis NEM760 was isolated from a stool swab collected on admission from a patient as surveillance for vancomycin-resistant enterococci. Strain NEM760 was identified as S. bovis by conventional biochemical methods and partial sequence analysis of its 16S rRNA. This strain was resistant to a low level of vancomycin (MIC, 64 micrograms/ml) but was susceptible to teicoplanin (MIC, 1 micrograms/ml), and vancomycin induced resistance to both glycopeptides. The presence of a vanB-related gene in NEM760 was demonstrated in a PCR assay which enabled specific amplification of a 635-hp internal segment of vanB. Sequence analysis of the corresponding PCR product revealed that it was highly homologous (96% identity) to the prototype vanB sequence of Enterococcus faecalis V583. The VanB resistance of determinant of S. bovis NEM760 was transferred by conjugation to E. faecalis and Enterococcus faecium at a similar frequency of 2 x 10(-5) per donor. SmaI-digested genomic DNAs of independently obtained transconjugants of E. faecalis and E. faecium were analyzed by pulsed-field gel electrophoresis and Southern hybridization with a vanB DNA probe. The electrophoretic and hybridization patterns obtained with all transconjugants of the same species were indistinguishable and revealed vanB-containing chromosomal insertions of approximately 100 kb. These results suggest that the genes mediating VanB-type resistance in S. bovis NEM760 are part of large transferable genetic elements. The results presented in the report demonstrate for the first time the role of streptococci in the dissemination of vancomycin resistance among gram-positive bacteria.

Congenital urethral obstruction: Cobb's collar or prolapsed congenital obstructive posterior urethral membrane (COPUM).

OBJECTIVE: To determine whether there are two distinct types of congenital obstruction of the proximal urethra. PATIENTS AND METHODS: Three boys with a congenital bulbar urethral narrowing (Cobb's collar) were examined. Recent papers on congenital obstructive posterior urethral membrane (COPUM) were reviewed. RESULTS: The more proximal lesion is a membranous obstruction which is able to prolapse as far as the bulbar urethra, but has paramedian folds that attach along the posterior wall of the urethra to the verumontanum, and is due to persistence of an embryological attachment between the distal verumontanum and the anterior wall of the posterior urethra. The more distal narrowing is not always obstructive and is primarily a bulbar urethral membrane, is independent of the verumontanum and external sphincter, and may represent a persistence of part of the urogenital membrane. CONCLUSIONS: There are two distinct types of congenital obstruction of the proximal urethra, with the association to the verumontanum being the distinguishing feature.

The common bilio-pancreatic channel syndrome in childhood.

An abnormally long common bilio-pancreatic channel has been found in association with choledochal cysts and biliary strictures in childhood. It may also present with recurrent abdominal pain, vomiting, hyperamylasaemia and jaundice. This has been termed the common channel syndrome (CCS). Two cases with the CCS presenting early in childhood are reported together with a review of the literature. Open sphincteroplasty was performed in both cases with a satisfactory outcome.

Ultrasound diagnosis of nephrocalcinosis in preterm infants.

The incidence of nephrocalcinosis in very low birthweight (less than 1500 g) premature infants was assessed by ultrasound scan and analysis of urine. Three of 36 infants had nephrocalcinosis. All had been receiving long term frusemide for bronchopulmonary dysplasia with simultaneous fluid restriction. Urinary investigations showed no consistent findings in babies with nephrocalcinosis.