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PubMed2002

Rapid identification of Candida species by confocal Raman microspectroscopy.

Candida species are important nosocomial pathogens associated with high mortality rates. Rapid detection and identification of Candida species can guide a clinician at an early stage to prescribe antifungal drugs or to adjust empirical therapy when resistant species are isolated. Confocal Raman microspectroscopy is highly suitable for the rapid identification of Candida species, since Raman spectra can be directly obtained from microcolonies on a solid culture medium after only 6 h of culturing. In this study, we have used a set of 42 Candida strains comprising five species that are frequently encountered in clinical microbiology to test the feasibility of the technique for the rapid identification of Candida species. The procedure was started either from a culture on Sabouraud medium or from a positive vial of an automated blood culture system. Prior to Raman measurements, strains were subcultured on Sabouraud medium for 6 h to form microcolonies. Using multivariate statistical analyses, a high prediction accuracy (97 to 100%) was obtained with the Raman method. Identification with Raman microspectroscopy may therefore be significantly faster than identification with commercial identification systems that allow various species to be identified and that often require 24 to 48 h before a reliable identification is obtained. We conclude that confocal Raman microspectroscopy offers a rapid, accurate, and easy-to-use alternative for the identification of clinically relevant Candida species.

Algorithms↗
PubMed2003

Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species.

BACKGROUND: The importance of identifying the pathogenic fungi rapidly has encouraged the development of differential media for the presumptive identification of yeasts. In this study two differential media, CHROMagar Candida and bismuth sulphite glucose glycine yeast agar, were evaluated for the presumptive identification of yeast species. METHODS: A total number of 270 yeast strains including 169 Candida albicans, 33 C. tropicalis, 24 C. glabrata, 18 C. parapsilosis, 12 C. krusei, 5 Trichosporon spp., 4 C. kefyr, 2 C. lusitaniae, 1 Saccharomyces cerevisiae and 1 Geotrichum candidum were included. The strains were first identified by germ tube test, morphological characteristics on cornmeal tween 80 agar and Vitek 32 and API 20 C AUX systems. In parallel, they were also streaked onto CHROMagar Candida and bismuth sulphite glucose glycine yeast agar plates. The results were read according to the color, morphology of the colonies and the existance of halo around them after 48 hours of incubation at 37 degrees C. RESULTS: The sensitivity and specificity values for C. albicans strains were found to be 99.4, 100% for CHROMagar Candida and 87.0, 75.2% for BiGGY agar, respectively. The sensitivity of CHROMagar Candida to identify C. tropicalis, C. glabrata and C. krusei ranged between 90.9 and 100% while the specificity was 100%. The sensitivity rates for BiGGY agar were 66.6 and 100% while the specificity values were found to be 95.4 and 100% for C. tropicalis and C. krusei, respectively. CONCLUSIONS: It can be concluded that the use of CHROMagar Candida is an easy and reliable method for the presumptive identification of most commonly isolated Candida species especially C. albicans, C. tropicalis and C. krusei. The lower sensitivity and specificity of BiGGY agar to identify commonly isolated Candida species potentially limits the clinical usefulness of this agar.

Journal Article↗
PubMed1985

Biochemical identification and phylogenetic relationships in free-living amoebas of the genus Naegleria.

Using isoelectric focusing, the zymograms of 23 pathogenic and nonpathogenic Naegleria strains were studied for the activity of 16 enzymes. Certain enzymes (lactate dehydrogenase, L-threonine dehydrogenase, superoxide dismutase, acid phosphatase, malic enzyme, and leucine aminopeptidase) proved particularly useful from a practical point of view as they allow easy and reliable identification of pathogenic N. fowleri and N. australiensis as well as nonpathogenic N. lovaniensis strains. Genetic interpretation of these zymograms gave estimates of genetic distances that largely confirmed the taxonomic position of the Naegleria species. In addition, the genetic data suggest that there are two main phylogenetic groups in the genus Naegleria.

Amoeba↗
PubMedDate not supplied

Viral pathogens of acute lower respiratory infections in pre-school Nigerian children and clinical implications of multiple microbial identifications.

In a prospective study of acute lower respiratory infections (ALRI), at the University College Hospital, UCH, Ibadan, 35 viral pathogens were identified by immunofluorescence (IF) techniques from 24 (68.6%) respiratory specimens from 35 hospitalised pre-school children. The respiratory diagnoses comprised croup, bronchiolitis, pneumonia and pleural effusion. The viral identifications comprised 14 (40.0%) of parainfluenza virus type 3, 10 (28.6%) of respiratory syncytial virus (RSV), 5 (14.3%) of influenza virus type A, 4 (11.4%) of parainfluenza virus type 1 and 2 (5.7%) of influenza virus type B. Two or more viral agents were identified in as many as 10 (41.7%) of the 24 IF positive secretions, 8 (80.0%) of which were obtained from children with features of protein energy malnutrition. Twenty subjects had both virological and bacteriological analyses, in 8 (40.0%) of whom co-existing bacteraemia was identified. Four (50.0%) of these blood culture positive subjects, also had features of overt malnutrition. Neither the age nor the sex was significantly related to the viral identifications (P > 0.81 & 0.35 respectively). Similarly, the final respiratory diagnoses were not significantly related to the viral identifications despite the seemingly suggestive relationship between a diagnosis of croup and parainfluenza identifications as well as that between pneumonia and RSV/parainfluenza type 3 identifications. It is concluded that the high proportion of positive viral identifications is a pointer to the importance of viruses as possible primary etiological agents of ALRI in countries of the West African sub-region and perhaps in developing countries of other tropical subregions. The multiplicity of microbial identifications (viruses and bacteria), seen in malnourished children, may explain the clinical severity of ALRI in the same group of children. The usefulness of IF as a rapid diagnostic tool, as well as the potential implications of our findings on ARI control in developing countries, are discussed.

Acute Disease↗
PubMed2005

Microarray analysis of Escherichia coli O157:H7.

AIM: To establish the rapid, specific, and sensitive method for detecting O157:H7 with DNA microchips. METHODS: Specific oligonucleotide probes (26-28 nt) of bacterial antigenic and virulent genes of E. coli O157:H7 and other related pathogen genes were pre-synthesized and immobilized on a solid support to make microchips. The four genes encoding O157 somatic antigen (rfbE), H7 flagellar antigen (fliC) and toxins (SLT1, SLT2) were monitored by multiplex PCR with four pairs of specific primers. Fluorescence-Cy3 labeled samples for hybridization were generated by PCR with Cy3-labeled single prime. Hybridization was performed for 60 min at 45 degrees. Microchip images were taken using a confocal fluorescent scanner. RESULTS: Twelve different bacterial strains were detected with various combinations of four virulent genes. All the O157:H7 strains yielded positive results by multiplex PCR. The size of the PCR products generated with these primers varied from 210 to 678 bp. All the rfbE/fliC/SLT1/SLT2 probes specifically recognized Cy3-labeled fluorescent samples from O157:H7 strains, or strains containing O157 and H7 genes. No cross hybridization of O157:H7 fluorescent samples occurred in other probes. Non-O157:H7 pathogens failed to yield any signal under comparable conditions. If the Cy3-labeled fluorescent product of O157 single PCR was diluted 50-fold, no signal was found in agarose gel electrophoresis, but a positive signal was found in microarray hybridization. CONCLUSION: Microarray analysis of O157:H7 is a rapid, specific, and efficient method for identification and detection of bacterial pathogens.

Escherichia coli Infections↗
PubMedDate not supplied

Pyrolysis mass spectrometry characterisation and numerical taxonomy of Aeromonas spp.

Reference strains (2) and 29 isolates of Aeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of 'typical' isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate between A. caviae and A. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis for A. caviae and A. sobria, but the main group of A. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate between A. hydrophila and A. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains of A. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable non-pathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.

Aeromonas↗
PubMed2003

Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR.

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the detection and identification of organisms of clinical and epidemiologic importance. Staphylococcus aureus, one of the most frequent causes of human infections, was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. One hundred clinical isolates of S. aureus, verified by biochemical reactions and latex agglutination and 90 negative control clinical isolates were screened in the assay. Moreover, fifty blood broth samples from blood culture bottles showing Gram-positive cocci in clusters on direct Gram's stain and 25 showing Gram-negative bacilli were screened. The probes, constructed from the nuc gene, correctly detected all S. aureus genomes present without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from other types of clinical specimens.

Base Sequence↗
PubMed2006

Severe skin and soft tissue infections and associated critical illness.

Skin and soft tissue infections (SSTIs) span a broad spectrum of clinical entities from limited cellulitis to rapidly progressive necrotizing fasciitis, which may be associated with septic shock or a toxic shock-like syndrome. These infections may manifest initially as pyodermas that then progress; alternatively, they may arise from metastatic spread of microorganisms from a distant focus. Regardless of the source, SSTIs may lead to critical illness. The complex interplay of environment, host, and pathogen are important to consider when evaluating SSTIs and planning appropriate therapy. The keys to a successful outcome are early identification of risk factors for specific pathogens and early initiation of empiric antimicrobial therapy. For certain types of SSTIs, surgical intervention for diagnosis and/or therapy is also required.

Journal Article↗
PubMedDate not supplied

[Identification of Escherichia coli and coliforms in childhood urinary sepsis using CROMOCEN CC chromogenic-fluorogenic medium].

In the last few years, the use of chromogenic and fluorogenic reactions for the identification of the most important pathogens in human urinary tract infections has became a powerful tool in clinic diagnostic. The study was carry out in two hospitals in Havana City, comparing the performance of experimental medium with the traditional one: C.L.E.D. Medium produced by Centro Nacional de Biopreparados (BIOCEN). Additional biochemical test were applied (indole, motility, citrate, H2S glucose and lactose) for the more accurate identification of different strains. During the test 119 positive samples were evaluated with 82 identified as E. coli (68.9%). As coliforms 18 samples (15.1%) were identify without any biochemical test. The diagnostic sensitivity was 100% and diagnostic specificity of new medium was of 97.4% for all assayed samples.

Bacteriological Techniques↗
PubMed2001

New (and used) approaches to the study of fungal pathogenicity.

The fungi are the most economically important plant pathogens and continue to be the focus of extensive research with a wide variety of methodologies. Enhancements in microscopy techniques have increased our ability to visualize the intimate interaction of fungi and their host plants. Improving methods allow pharmacological inhibition and genetic dissection of the determinants of fungal pathogenicity in a gene-by-gene approach. Identification and analysis of genes differentially transcribed in ways pertinent to pathogenicity continues to be a frequent research approach. Genome-wide analysis is gaining favor in biological research and fungal plant pathogens are no exception. Several industrial research groups are exploring fungal plant pathogenesis based on genomic sequence data and genome-wide mutagenesis. In March 2001 the first publicly available complete genome of a filamentous fungus (Neurospora crassa) was released. N. crassa is of course a saprophyte and there is no complete sequence available for a plant pathogenic fungus in public databases. However, freely accessible entire genome sequences for both plant pathogenic fungi and their hosts are on the horizon. Sequence availability promises to revolutionize the rate at which data relevant to disease processes will be accrued. In this review we describe approaches currently applied to the study of plant pathogenic fungi and explore developments of potential future benefit with existing technologies not yet applied to this group of important organisms.

Fungi↗
PubMed2005

Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics.

The occurrence of the two Shewanella species found in clinical specimens, Shewanella algae and Shewanella putrefaciens, correlates with the temperature and salinity of seawater. This means that Shewanella infections occur in warm climates or during especially warm summers in temperate climates. The infections described most commonly involve ears, skin and soft tissue, with or without bacteraemia. Primary bacteraemia with a fulminant course is also seen in immunocompromised patients. Important differential characteristics between the two species include the ability of S. algae to produce mucoid colonies with beta-haemolysis on sheep blood agar, to grow at 42 degrees C and in NaCl 6% w/v, and to reduce nitrite, and an inability to produce acid from maltose, all of which are in contrast to the characteristics of S. putrefaciens. Automated identification systems fail to differentiate between S. algae and S. putrefaciens, as S. algae is not included in the databases of these systems. Presumably for this reason, most Shewanella infections reported during recent years have been attributed to S. putrefaciens. However, when extensive phenotypic characterisation is performed, most human infections are seen to be caused by S. algae. As the two species seem to have different pathogenic potential for humans, correct identification is important, and this is possible in routine clinical microbiology laboratories.

Animals↗
PubMed2001

Identification of a new iron-regulated virulence gene, ireA, in an extraintestinal pathogenic isolate of Escherichia coli.

Our laboratory is studying an extraintestinal pathogenic isolate of Escherichia coli (CP9) as a model pathogen. We have been using human urine, ascites, and blood ex vivo to identify genes with increased expression in these media relative to expression in Luria-Bertani (LB) broth. Such genes may represent new or unrecognized virulence traits. In this study, we report the identification of a new gene, ireA (iron-responsive element). This gene has an open reading frame of 2,049 nucleotides, and its peptide has a molecular mass of 75.3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its expression is increased a mean of 3.6-fold in human urine, 16.2-fold in human ascites, and 6.6-fold in human blood relative to expression in LB medium, and it is Fe repressible. IreA also exhibits peptide similarities (48 to 56%) to previously identified proteins that function as siderophore receptors, suggesting that IreA is involved in iron acquisition. PCR-based analysis of ireA's phylogenetic distribution detected ireA in none (0%) of 14 fecal isolates that represented probable commensal strains, but in 13 (26%) of 50 random urine and blood clinical isolates (P = 0.05) and in 5 (100%) of 5 representatives of the J96-like, clonal group of which CP9 is a member (P < 0.001). In a mouse urinary tract infection model, the presence of ireA contributed significantly to CP9's ability to colonize the bladder (P < 0.02), evidence that IreA is a urovirulence factor. Taken together, these findings demonstrate that ireA encodes a new virulence factor, which is likely involved in Fe acquisition.

Animals↗
PubMed2004

Familial myopathy: new insights into the T14709C mitochondrial tRNA mutation.

We have defined the genetic defect in a large family first described in one of the earliest reports of suspected mitochondrial myopathy, as the mutation T14709C in the mitochondrial transfer RNA(Glu) (mt-tRNA(Glu)) gene. Extraordinarily, this mutation has attained homoplasmy (100% mutated mt-tRNA(Glu)) on at least three independent occasions in this family and has done so in one individual who remains asymptomatic with no clinical evidence of disease. Heteroplasmy (dual populations of mutated and wild-type mtDNA) usually is regarded as one of the primary diagnostic criteria for pathogenicity and previous reports of the T14709C mutation detail heteroplasmy in a variety of tissues. In contrast, homoplasmy of mt-tRNA mutations generally has been regarded as evidence of a benign nature, with rare exceptions that result in organ-specific phenotypes. Discovering that T14709C, a common and severe mt-tRNA mutation, can attain homoplasmy without symptoms or clinical signs of disease has profound implications for the identification and prevalence of other pathogenic mt-tRNA mutations. Furthermore, variation in phenotype between homoplasmic individuals implies a crucial contribution from the nuclear genetic environment in determining the clinical outcome of mt-tRNA mutations.

Adolescent↗
PubMed2004

A Reed-Frost model taking into account uncertainties in the diagnostic of the infection.

In this paper, we model the epidemic course of a pathogen infection within a semi-closed group which generates clinical signals which do not necessarily permit its ready and certain identification. Typical examples of such a pathogen are influenza-type viruses. We allow for time-varying infectivity levels among individuals, and model the probability of infection per contact as a function of the clinical signals. In order to accomplish this, we introduce a modified chain-binomial Reed-Frost model. We obtain an expression for the basic reproduction ratio and determine conditions which guarantee that the epidemic does not survive in the long-term. These conditions being functions of the signal's distribution, they can be used to design and evaluate interventions, such as treatment protocols.

Disease Outbreaks↗
PubMed2004

[The novel non-A, non-E hepatitis viruses and their pathogenic effect].

Molecular techniques have allowed the identification of new viruses in a number of patients with cryptogenic hepatitis. Whether they are clinically inapparent or true hepatitis agents remains unknown for some of them. Latest described viruses include GBV, TTV and SENV. However, based on the limited data available, they do not seem to be contenders for the new hepatitis virus title. However, researchers are looking for a role of these viruses in other chronic and acute human diseases. Only a careful evaluation of the data and the scientific concordance of all the evidence will resolve the question of whether they are only commensal viruses or pose a real pathogenic potential.

Circoviridae↗
PubMed2004

[Rapid identification of Candida glabrata using a new commercial kit].

Candida glabrata is an emergent pathogen with diminished susceptibility to azoles, thus a rapid identification of this yeast could be of help to choose the appropriate treatment. GLABRATA RTT (Fumouze Diagnostics, France) is a new C. glabrata identification test. To evaluate its utility in the clinical laboratory daily routine, we prospectively tested 168 yeasts isolated in our hospital. GLABRATA RTT results had a sensitivity of 98.4% and a specificity of 100%. The combination of CHROMagar Candida isolation medium and GLABRATA RTT test allowed the identification of the four most common species in the clinical practice (Candida albicans, Candida tropicalis, Candida krusei and C. glabrata).

Candida glabrata↗
PubMed1992

Comparative evaluation of a chemiluminescent DNA probe and an exoantigen test for rapid identification of Histoplasma capsulatum.

A chemiluminescent DNA probe (Accuprobe) assay developed by Gen Probe, Inc., for the rapid identification of Histoplasma capsulatum was evaluated and compared with the exoantigen test by using 162 coded cultures including Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farciminosum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and morphologically related saprobic fungi. Each test uses a chemiluminescent, acridinium ester-labeled, single-stranded DNA probe that is complementary to the rRNA of the target organism. Lysates of the test cultures were prepared by sonication with glass beads and heat treated. After the rRNA was released from the target organism, the labeled DNA probe combined with the target H. capsulatum rRNA to form a stable DNA-RNA hybrid. A hybridization protection assay was used, and the chemiluminescence of hybrids was measured initially with a Leader 1 luminometer as relative light units and later during the investigation with a probe assay luminometer as probe light units. Of the 162 coded mycelial cultures tested by the Accuprobe assay, 105 were identified as H. capsulatum. The test could be performed with an inoculum of a few square millimeters (1 to 2 mm2) of growth. In the primary evaluation, the Accuprobe identified 103 of the 105 cultures as H. capsulatum within 2 h. The remaining two cultures, contaminated with bacteria, had to be purified before the Accuprobe assay identified them correctly as H. capsulatum. Since each coded culture was concurrently tested for H. capsulatum, B. dermatitidis, and C. immitis exoantigens, the identification of all three dimorphic pathogens was provided simultaneously. Of the 162 coded cultures tested, 105 were identified by the exoantigen test as H. capsulatum, 12 were identified as B. dermatitidis, 13 were identified as C. immitis, and 32 were negative for H. capsulatum, B. dermatitidis, and C. immitis. The bacterial contamination in two isolates did not interfere with the exoantigen testing. The exoantigen test required 7- to 10-day-old colonies and required 48 to 72 h of incubation before definitive identification was obtained.

Antigens, Fungal↗
PubMed1975

[A method for routine identification of anaerobic bacteria, in particular of non-sporogenic forms (author's transl)].

In the Institute of Bacteriology of Allgemeines Krankenhaus St. Georg, Hamburg, all clinical specimens received (except stools, sputum, and vaginal swabs) have been studied for the presence of anaerobians since 1947. More than 25 years' experience has convinced the authors that not infrequently, non-sporogenic anaerobians occur as infectious agents. Statistical data compiled for the period 1956-1972 revealed that from a total of 71,973 specimens contaminated with bacteria, 7,047 (9.8%) were containing non-sporogenic anaerobians. Pure anaerobic cultures were obtained in 2.404 cases (3.3%) and mixed cultures of aerobic and anaerobic bacteria in 4,643 cases (6.5%). The pathogenic importance was revealed by identifications from osteomyelitis cases: 11-13.7% pure cultures and 17% mixed cultures (with aerobians). Also in cases of total endoprosthesis of complicated course they were detected in up to 23%. On account of the frequent cultures of non-sporogenic anaerobians obtained from clinical specimens, the authors demand a routine analysis of all clinical specimens for the presence of anerobians...

Agar↗