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Diagnosis of necrotizing fasciitis in children.

Necrotizing fasciitis is a rare but progressive soft tissue infection. This condition is difficult to recognize in the early phase, when it is often confused with cellulitis. We report the cases of four children with necrotizing fasciitis. The initial presentation in these cases was cellulitis. Fever and soft tissue swelling occurred within 24 h and spreading erythema within 4 to 12 h. Radiologic studies of the lesions showed soft tissue thickening. Ultrasonography of the lesions demonstrated distorted, thickened fascia with fluid accumulation. Well-defined, loculated abscesses were demonstrated in two cases. Although typical dusky skin and purplish patches were not found in our cases, necrotizing fasciitis was strongly suspected on the basis of the clinical course and sonographic findings. Ultrasonography also was used as a guide for aspiration of pus. Gram-stained smears and bacterial cultures yielded the pathogens. The choice of antibiotic therapy was made on the results of smears and culture. All patients survived after immediate surgical debridement, intensive antibiotic therapy, and aggressive wound care. In conclusion, ultrasonography provides a rapid and valuable diagnostic modality for necrotizing fasciitis. The pus obtained through sonographically guided aspiration for bacterial culture can allow identification of the pathogenic organisms.

Adolescent↗

Prospective multicenter study of the causative organisms of community-acquired pneumonia in adults in Japan.

Although a wide variety of recognized pathogens causes community-acquired pneumonia, the precise etiology in Japan remains unknown. We prospectively investigated the etiology in 232 patients with community-acquired pneumonia who visited 20 community-general hospitals. New diagnostic methods, using polymerase chain reaction (PCR) assays and urinary antigen tests were employed, in addition to conventional methods. The frequency of identification of causative pathogens was high (73.3%), and the leading organism was Streptococcus pneumoniae (24.6%), followed by Haemophilus influenzae (18.5%), viruses (16.4%), Chlamydia pneumoniae (6.5%), Mycoplasma pneumoniae (5.2%), and Legionella spp. (3.9%). S. pneumoniae and M. pneumoniae were the most prevalent pathogens in younger patients, and S. pneumoniae and H. influenzae were the most prevalent in elderly patients. Multiple or mixed infections were found in 25.9% of all patients and in 35.3% with a causal diagnosis. The results have important practical implications for the initial treatment of adult patients with community-acquired pneumonia.

Adolescent↗

[Antibiotic therapy of infectious endocarditis (when, with what drug, how long?].

The aim of antibiotic therapy in bacterial endocarditis is to sterilize infected cardiac structures and vegetations. Pathogenic organisms are present in great numbers within vegetations and abscess-formations. They exist in a state of reduced metabolic activity so that they are able to tolerate even therapeutic levels of bactericidal antibiotic concentrations. Because vegetations are normally devoid of blood vessels, impregnation with antibiotic agents is poor. Effective therapy is greatly improved by identification of the pathogenic organism involved. With very few exceptions isolation is possible prior to initiation of antibiotic therapy. In acute cases with signs of septicemia, however, therapy cannot await results of bacterial testing. In these patients selection of antibiotic agents is based on associated evidence such as the presence of a prosthetic heart valve or intravenous drug addiction. Once the pathogen has been identified antibacterial therapy should be tailored according to the test results. Bactericidal antibiotics should always be preferred over bacteriostatic agents; in many cases adequate bactericidal levels can only be achieved by combining various agents, such as ampicillin and gentamycin for treatment of enterococcal endocarditis. Dosing intervals must take into account the resulting trough levels, which should always exceed the minimal inhibitory concentrations for a specific bacterial strain. In cases with inadequate control of infection, congestive heart failure resulting from valve dysfunction, and abscess formation, surgery as the only means of eradicating the infection and restoring cardiac performance should not be delayed.

Anti-Bacterial Agents↗

Genetic heterogeneity of the pathogenic potentials of human and bovine group B streptococci.

One-hundred seventy-two B-streptococcal strains of human and bovine origin were analyzed for the presence of 9 genes potentially involved in virulence. Some of genes (glnA, cyl, hylB, scaA and cfb) were revealed in all the strains. However, the presence of others (bca, bac, scpB, lmb) varied from strain to strain. Taken together, 3 and 5 different types of pathogenic potential were found among human and bovine group B streptococci (GBS) strains, respectively, and only one type (bca+ bac scpB+ glnA+ cyl+ hylB+ lmb+ scaA+ cfb+) was common for both kinds of strains. We propose that different virulence genes can be involved in the development of infectious processes in humans and animals. A reliable PCR protocol with 3 pairs of primers (for the genes bca, bac and scpB) in the same reaction mixture was developed for the fast identification of the pathogenic potential of GBS. In comparison with the classical immunological methods this procedure displayed higher specificity and sensitivity as well as a shorter time of analysis. It can be recommended for use in the clinical and veterinary practice for studying the epidemiological relationship between the isolates and the ready identification of the clone causing the infection.

Animals↗

Microbial invasion of the amniotic cavity at birth is associated with adverse short-term outcome of preterm infants.

AIMS: To determine the frequency and clinical significance of microbial invasion of the amniotic cavity at the time of delivery in preterm infants. METHODS: Prospective cohort study during June 2001 and January 2002. Preterm infants < 33 + 6 weeks of gestation who had amniotic fluid and placental tissue sampled for culture during cesarean section were included. RESULTS: Of a total of 80 neonates, 42 had negative culture results, 22 had growth of Ureaplasma urealyticum, and 16 had growth of other pathogens. Isolation of Ureaplasma urealyticum was associated with a decreased risk of developing hyaline membrane disease after birth but a more than 20 times increased risk of developing chronic lung disease. Patients with growth of other pathogens had a significantly higher mortality than patients with negative culture results. CONCLUSIONS: Isolation of miroorganisms from the amniotic cavity at birth is associated with an adverse outcome of the preterm infant. In the light of extremely small numbers of positive blood cultures in preterm infants after birth, we consider it reasonable to recommend routine culturing of amniotic cavity tissues/fluid obtained during cesarean section in order to increase the identification rate of pathogens potentially involved in the pathogenesis of perinatal infections.

Chorioamnionitis↗

Specific oligonucleotide primers for identification of Cladophialophora carrionii, a causative agent of chromoblastomycosis.

Cladophialophora carrionii is one of the relatively common causative agents of chromoblastomycosis. We have developed the specific oligonucleotide primer set based on the internal transcribed spacer regions of ribosomal DNA for the rapid identification of this pathogen. PCR with this primer set amplified a 362-bp amplicon from C. carrionii strains. From other relevant dematiaceous species, including medically important dematiaceous fungi, such as Fonsecaea pedrosoi, Phialophora verrucosa, and Exophiala dermatitidis, and eight species of medically important yeasts, such as Candida albicans and Cryptococcus neoformans var. neoformans, the primer set did not produce any amplicon. PCR with this primer set may be a useful tool for the identification of C. carrionii.

Chromoblastomycosis↗

[Risk factors for persistent diarrhea].

The current concepts in persistent diarrhoea (PD) are reviewed. 102 patients with acute diarrhoea and 85 with PD are studied. All were less than one year old. Information about age, nutritional status, time of breast feeding, previous episodes of acute and persistent diarrhoea and the previous use of drugs is registered. Microbiological and parasitologics studies were done to identify enteric pathogens. To prove the association of PD and the exposure to different risk factors the statistic method of Chi square (chi 2) was used and the odds ratio (OR) estimated. In order of importance and according to the OR the risk factors identified were: previous episodes of PD, malnutrition, less than one month with breast feeding, the use of metronidazol and antibiotics, multiple infections, previous episodes of acute diarrhoea and the identification of enteric pathogens. The most frequent pathogen was Salmonella followed by entero pathogenic Escherichia coli.

Acute Disease↗

Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf coast molluscan shellfish at harvest.

The densities of total and pathogenic Vibrio parahaemolyticus in 671 samples of molluscan shellfish harvested in 1999 and 2000 from 14 sites in seven Gulf and Atlantic coast states were determined at 2-week intervals over a period of 12 to 16 months in each state. Changes in V. parahaemolyticus densities in shellfish between harvest and sample analysis were minimized with time and temperature controls. Densities were measured by direct plating techniques, and gene probes were used for identification. Total and pathogenic V. parahaemolyticus organisms were identified with probes for the thermolabile direct hemolysin (tlh) gene and the thermostable direct hemolysin (tdh) gene, respectively. An enrichment procedure involving 25 g of shellfish was also used for the recovery of pathogenic V. parahaemolyticus. The densities of V. parahaemolyticus in shellfish from all harvest sites were positively correlated with water temperature. Shellfish from the Gulf Coast typically had higher densities of V. parahaemolyticus than did shellfish harvested from the North Atlantic or mid-Atlantic coast. Vibrio parahaemolyticus counts exceeded 1,000 CFU/g for only 5% of all samples. Pathogenic (tdh+) V. parahaemolyticus was detected in approximately 6% of all samples by both procedures, and 61.5% of populations in the positive samples from the direct plating procedure were at the lower limit of detection (10 CFU/g). The frequency of detection of pathogenic V. parahaemolyticus was significantly related to water temperature and to the density of total V. parahaemolyticus. The failure to detect pathogenic V. parahaemolyticus in shellfish more frequently was attributed to the low numbers and uneven distribution of the organism.

Animals↗

Separation of sequences from host-pathogen interface using triplet nucleotide frequencies.

The identification of genes involved in host-pathogen interactions is important for the elucidation of mechanisms of disease resistance and host susceptibility. A traditional way to classify the origin of genes sampled from a pool of mixed cDNA is through sequence similarity to known genes from either the pathogen or host organism or other closely related species. This approach does not work when the identified sequence has no close homologues in the sequence databases. In our previous studies, we classified genes using their codon frequencies. This method, however, explicitly required the prediction of CDS regions and thus could not be applied to sequences composed from the non-coding regions of genes. In this study, we show that the use of sliding-window triplet frequencies extends the application of the algorithm to both coding and non-coding sequences and also increases the prediction accuracy of a Support Vector Machine classifier from 95.6+/-0.3 to 96.5+/-0.2. Thus the use of the triplet frequencies increased the prediction accuracy of the new method by more than 20% compared to our previous approach. A functional analysis of sequences detected gene families having significantly higher or lower probability to be correctly classified compared to the average accuracy of the method is described. The server to perform classification of EST sequences using triplet frequencies is available at (URL: http://mips.gsf.de/proj/est3).

Algorithms↗

Nucleic Acid-based Pathogen Discovery Techniques: Potential Application to Xenozoonoses.

Pathogen discovery techniques based on detection and characterization of microbial nucleic acids have potential application to the field of xenotransplantation. Techniques such as broad-range polymerase chain reaction and representational difference analysis may make it possible to define the zoonotic flora present within donor animal species to an extent never before possible. This review describes the current practice of these techniques, with an emphasis on the use of these methods for identifying zoonotic agents. Identification of potential pathogens in colony-reared donors may lead to isolation and selective breeding practices that could ultimately eliminate these agents from the donor pool.

Journal Article↗

[Diagnosis and treatment of brain abscesses].

The etiologic pathogens of brain abscesses vary depending on the underlying disease. Aerobic and anaerobic bacteria are frequently involved simultaneously. In most cases, the clinical course is subacute. C-reactive protein is the most sensitive inflammatory parameter in the blood. It is elevated in 80 to 90% of all cases. The diagnosis is made by cranial computer tomography without and with contrast enhancement. The rapid culture of pus from the abscess cavity is crucial for the identification of the pathogen. Antibiotic therapy alone is indicated 1. in the presence of multiple, small and/or deep-seated abscesses or 2. when the general condition of the patient does not allow surgery at an acceptable risk or 3. in early cerebritis without capsule formation. Frequently used surgical procedures are abscess aspiration (usually by stereotaxic surgery), open craniotomy and excision of the abscess with the capsule, and open evacuation of the abscess cavity. For empirical treatment the combination of cefotaxime (3 x 2-4 g/d i.v.) plus metronidazol (3-4 x 0.5 g/d i.v.) is preferred. Corticosteroids are indicated in the presence of a space-occupying effect and imminent brain herniation, or of multiple abscesses and abscesses in critical brain regions such as in the cerebellum.

Anti-Bacterial Agents↗

Corneal biopsy in chronic keratitis.

Failure to identify the causative agent makes the treatment of chronic keratitis difficult. Inability to obtain a definitive diagnosis may be related to the depth of the pathogen within the cornea, partial or inappropriate treatment that interferes with diagnostic cultures, or fastidious growth characteristics of the organism in culture. Biopsy by lamellar keratectomy provides sufficient tissue for culture and for histopathologic evaluation by histochemistry, immunohistopathology, and, if necessary, electron microscopy. Consultation with an ophthalmic pathologist before biopsy can prevent inappropriate handling of the specimen, which may preclude essential diagnostic procedures. Many studies can be done within 24 hours of biopsy, often permitting rapid identification of the pathogen and prompt institution of appropriate therapy.

Biopsy↗

Selective procedure to isolate haemophilus influenzae from sputa with large quantities of Pseudomonas aeruginosa.

The identification of respiratory pathogens (e. g. Haemophilus influenzae, Streptococcus pneumoniae) is impaired by the presence of large quantities of Pseudomonas aeruginosa, as is the case in the sputum specimens of cystic fibrosis patients. A procedure has been evaluated whereby the selective inhibition of the proliferation of P. aeruginosa is achieved by a broad spectrum pyocin, whereas the growth of H. influenzae is not influenced. This technique has been tested over a two year period resulting in a significantly augmented rate of identification of H. influenzae.

Bacteriocins↗

Identification of the mycobacterial carbohydrate structure that binds the C-type lectins DC-SIGN, L-SIGN and SIGNR1.

Mycobacterium tuberculosis represents a worldwide health risk and although macrophages are primarily infected, dendritic cells (DC) are important in inducing cellular immune responses against M. tuberculosis. Recent studies have demonstrated that M. tuberculosis targets the DC-specific C-type lectin DC-SIGN to inhibit the immuno-stimulatory function of DC through the interaction of the mycobacterial mannosylated lipoarabinomannan (ManLAM) to DC-SIGN, which prevents DC maturation and induces the immuno-suppressive cytokine IL-10. This may contribute to survival and persistence of M. tuberculosis. Here, we have identified the specific pathogen-derived carbohydrate structure on ManLAM that is recognized by DC-SIGN. We have synthesized the mannose-cap oligosaccharides man-ara, (man)2-ara and (man)3-ara, and demonstrate that these neoglycoconjugates are specifically bound by DC-SIGN. Moreover, we demonstrate that the human and murine DC-SIGN homologue L-SIGN and SIGNR1, respectively, also interact with mycobacteria through ManLAM. Both homologues have the highest affinity for the (man)3-ara structure, similar to DC-SIGN. This study provides information about the specific carbohydrate structures on pathogens that are recognized by DC-SIGN, and may provide strategies to develop vaccines against these pathogens. Moreover, the identification of SIGNR1 as a receptor for ManLAM will enable in vivo studies to investigate the role of DC-SIGN in M. tuberculosis pathogenesis.

Animals↗

[Pathogenicity factors in Botrytis cinerea].

Botrytis cinereais an important plant pathogenic fungi with a wide host range, which can make use of different infection mechanisms. Although genetic variation for resistance to B. cinereahas been observed within some species, no gene-for-gene relationship has been found. The development of resistant genotypes is, therefore, complicated. Any attempt to develop control strategies makes it necessary a detailed knowledge of both the fungal infection mechanisms and the plant defence mechanisms. The application of different experimental approaches allows the analysis of the infection process in different hosts, the description of the elements that participate in each stage of the process and the identification of those pathogenicity factors which are essential for the establishment of the interaction. The characterisation of the latter will provide information about key elements of the infection process as the basis for the development of effective, long term and environmentally friendly control strategis.

English Abstract↗

Serotyping and ribotyping of Salmonella using restriction enzyme PvuII.

The subtyping and identification of bacterial pathogens throughout food processing and production chains is useful to the new hazard analysis critical control point-based food safety plans. Traditional manual serotyping remains the primary means of subtyping Salmonella isolates. Molecular biology techniques, however, offer the promise of more rapid and sensitive subtyping of Salmonella. This study evaluates the potential of restriction enzyme PvuII, followed by probing with the rRNA operon from Escherichia coli, to generate serotype-specific DNA fingerprints. A total of 32 identified serotypes were found with an overall agreement in 208 of the 259 (80%) isolates tested between U.S. Department of Agriculture serotype identification and riboprint serotype identification. Many of the isolates that did not correlate were serotype identified as Salmonella Montevideo, which indicates that for this serotype, there are multiple ribotypes. When Salmonella Montevideo isolates were not included, the ribotype identification agreed with serotyping in 207 of the 231 (90%) isolates. The primary outcome of any ribotyping procedure is to give distinct ribotype patterns. This extensive poultry epidemiological study demonstrates that, in addition to ribotype patterns, the identification of isolates to known serotypes provides the investigator with additional information that can be more useful than traditional epidemiology and isolate identification studies.

Animals↗

Identification of Acinetobacter isolates in the A. calcoaceticus-A. baumannii complex by restriction analysis of the 16S-23S rRNA intergenic-spacer sequences.

Members of the genus Acinetobacter are reported to be involved in hospital-acquired infections with an increasing frequency. However, clinical laboratories still lack simple methods that allow complete identification of some pathogenic species, i.e., those corresponding to A. baumannii (DNA group or genospecies 2), unnamed genospecies 3 and 13, and two new genospecies that have recently been described. In fact, a complete discrimination between these species is possible only by DNA-DNA hybridization or ribotyping. Both of these techniques are complex and time-consuming and cannot be performed in most clinical laboratories. As a consequence, isolates belonging to these genospecies are often not differentiated and included, together with the environmental genospecies 1, in the A. calcoaceticus-A. baumannii complex. In this report, a simple and rapid method for the identification of the genospecies belonging to the A. calcoaceticus-A. baumannii complex is proposed. It is based on the combined digestion by the restriction endonuclease AluI and NdeII of the DNA fragments resulting from the amplification of the 16S-23S rRNA intergenic spacer sequences. The analysis of 36 strains characterized by DNA-DNA hybridization in previous studies showed that the restriction profiles obtained are highly reproducible and characteristic for each genospecies. Moreover, extending this study to 68 clinical strains, which were assigned to the A. calcoaceticus-A. baumannii complex by phenotypic tests, confirmed the existence of a panel of limited and well-conserved restriction patterns and allowed the identification of the strains tested. This study thus proposes the detection of restriction length polymorphism in the spacer sequences between the 16S and 23S rRNA genes as a method for the identification of isolates in the A. calcoaceticus-A. baumannii complex.

Acinetobacter↗

Infections in febrile granulocytopenic patients: clinical features and pathogens.

From 1990 to 1994, we prospectively evaluated patients with cancer or aplastic anemia who had granulocyte counts of less than 500/mm3 and fever, in order to study infections in febrile granulocytopenic patients in Taiwan. A total of 100 episodes in 95 patients were evaluated. Aerobic Gram-negative bacilli were responsible for 72.5% of the 80 organisms identified in the infections. Escherichia coli was the most common isolate, accounting for 46.5% of Gram-negative bacilli. Pseudomonas aeruginosa and Klebsiella spp caused 24.1% and 18.9% of these infections, respectively. Aerobic Gram-positive cocci were responsible for 12.5% of the 80 organisms identified in the infections. Fungal infections were responsible for 8.8% of isolates. Septicemia, predominantly due to Gram-negative bacilli, accounted for 39 episodes. Infection sites included the respiratory tract, urinary tract, skin and soft tissue, oral cavity, intestines, anus and ear canal. Identification of the pathogens and their clinical features is important in the immediate treatment of such infections.

Adolescent↗