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PubMed2005

Identification of medically important molds by an oligonucleotide array.

Infections caused by fungi have increased in recent years. Accurate and rapid identification of fungal pathogens is important for appropriate treatment with antifungal agents. On the basis of the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 64 species (32 genera) of clinically important filamentous (or dimorphic) fungi. These 64 species included fungi causing superficial, cutaneous, subcutaneous, and invasive infections. The method consisted of PCR amplification of the ITS regions using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species- or group-specific oligonucleotides immobilized on a nylon membrane. Of 397 fungal strains (290 target and 107 nontarget strains) tested, the sensitivity and specificity of the array was 98.3% (285/290) and 98.1% (105/107), respectively. Misidentified strains were usually those belonging to the same genus of the target species or having partial homology with oligonucleotide probes on the membrane. The whole procedure can be finished within 24 h starting from isolated colonies; reproductive structures, which are essential for the conventional identification methods, are not needed. In conclusion, the present array is a powerful tool for identification of clinically important filamentous fungi and may have the potential to be continually extended by adding further oligonucleotides to the array without significantly increasing the cost or complexity.

Fungi↗
PubMed1986

Reliability of exoantigens for differentiating Blastomyces dermatitidis and Histoplasma capsulatum from Chrysosporium and geomyces species.

A recent study suggested that Chrysosporium species have the same diagnostic antigens as Histoplasma capsulatum and Blastomyces dermatitidis and, thus, compromise the antigenic identification of these pathogens. In light of these findings, studies were undertaken to determine the reliability of the exoantigen tests for identifying B. dermatitidis and H. capsulatum organisms from cultures. Sixty-three slant or shake culture extracts, or both, were derived from C. asperatum, C. keratinophilum, C. parvum, C. pruinosum, C. parvum var. crescens, Geomyces (Chrysosporium) pannorus, B. dermatitidis, and H. capsulatum. These were analyzed by use of a commercial exoantigen kit and exoantigen test reagents obtained from a commercial source. The results of these analyses were compared with those obtained with Centers for Disease Control reagents. Many of the extracts derived from nonpathogenic fungi produced nonspecific precipitin bands when reacted with the kit and reference antisera, particularly the B. dermatitidis antisera. None, however, produced antigens identical to the specific B. dermatitidis A and H. capsulatum H and M antigens. Our findings indicate that the properly controlled immunoidentification procedure is 100% specific for B. dermatitidis and H. capsulatum, and that cross-reacting antigens derived from morphologically similar saparophytic fungi do not pose identification problems.

Antigens, Fungal↗
PubMed1990

Phosphatase-novobiocin-mannose-inhibition test (PNMI-test) for routine identification of the coagulase-negative staphylococcal urinary tract pathogens S. epidermidis and S. saprophyticus.

A modified Kloos/Schleifer-scheme proved to be useful in identifying coagulase-negative staphylococci isolated from urine. S. epidermidis (44.2%) and S. saprophyticus (21.5%) were the most frequent species. Analysis of patients confirmed both species as urinary pathogens. Using an abbreviated scheme of 6 characteristics, S. saprophyticus was mis-classified in 19.5% of cases. A Phosphatase-Novobiocin-Mannose-Inhibition Test (PNMI-Test) together with a high NaCl concentration (10%) in combination with a coagulase test seems to be an acceptable compromise for routine identification of the three most important staphylococcal urinary tract pathogens, S. aureus, S. epidermidis, and S. saprophyticus. The technical and financial expenditure can be reduced considerably, because an extended identification has to be applied only to strains which cannot be identified by the PNMI-Test.

Bacteriuria↗
PubMed1999

Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene.

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10(5) by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity).

Animals↗
PubMed2006

Clinical implications of emerging pathogens in haemophilia: the variant Creutzfeldt-Jakob disease experience.

The impact of variant Creutzfeldt-Jakob disease (vCJD) on the clinical practice of haemophilia in the UK is coloured by the haemophilia community's experience of hepatitis C virus and human immunodeficiency virus (HIV) transmission via plasma-derived therapies in the 1980s, when the delay in recognizing and acting on the potential risks cost many patients their lives and left others to manage another chronic disease. This crisis prompted organisations such as the United Kingdom Haemophilia Centre Doctors' Organisation to advocate for the introduction of haemophilia therapies that would not be susceptible to contamination with blood-borne pathogens. After the identification of vCJD in 1996, a number of public health measures were taken in response to a government-sponsored vCJD risk assessment, and following reports of transfusion-transmission of vCJD, additional guidelines have been developed to prevent person-to-person transmission, some of which may impact the quality and availability of medical and surgical care. Variant CJD has had a significant negative effect on the UK haemophilia community, shaking patient confidence in the therapies they have received over the last 21 years, affecting the quality of care and creating the risk of stigmatizing the community as it was in the 1980s. As with HIV and vCJD, emerging blood-borne infectious agents will likely affect blood and blood-derived therapies well before we become aware of its presence. As a result, only therapies with the lowest level of risk should be used for care of patients with haemophilia.

Animals↗
PubMedDate not supplied

Extensive reprogramming of primary and secondary metabolism by fungal elicitor or infection in parsley cells.

The transcription rates of numerous plant genes have previously been shown to be strongly affected by pathogen infection or elicitor treatment. Here we estimate the extent and complexity of this response by analyzing the patterns of mRNA induction in fungal elicitor-treated parsley cells (Petroselinum crispum) for several representatives from various primary and secondary metabolic pathways, cytosolic as well as plastidic. As a reference, we use the biphasic accumulation curve for the coordinately induced mRNAs encoding the three core enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 4-coumarate:CoA ligase. Coincidence with this curve was observed for the mRNA induction kinetics of several, but not all, phenylpropanoid branch pathway-related reactions, whereas seven selected mRNAs from the pentose phosphate, glycolytic and shikimate pathways, including various cytosolic and plastidic isoforms, were induced with great differences in timing. Likewise unique and dissimilar from the reference curve were the induction patterns for various mRNAs encoding enzymes or proteins that are either more distantly or not at all related to phenylpropanoid metabolism. None of over 40 mRNAs tested so far remained unaffected. Using one strongly elicitor-responsive mRNA from carbohydrate metabolism, encoding a cytosolic glucose 6-phosphate dehydrogenase, for in situ RNA/RNA hybridization in fungus-infected parsley leaf tissue, we observed again the previously reported, close simulation of metabolic changes in true plant/fungus interactions by elicitor treatment of cultured cells. In addition to demonstrating extensive, highly complex functional, temporal and spatial patterns of changes in gene expression in infected plant cells, these results provide valuable information for the identification of pathogen-responsive promoters suitable for gene technology-assisted resistance breeding.

Apiaceae↗
PubMed2006

Identification of Vibrio isolates by a multiplex PCR assay and rpoB sequence determination.

Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus-account for the majority of Vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus). The assay was tested on a sample of 309 Vibrio isolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional four V. parahaemolyticus isolates among the other 119 isolates. Sequence analysis based on rpoB was used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticus sequences. The rpoB sequences for 12 of 15 previously unidentified isolates clustered with other Vibrio species in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibrio species. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibrio pathogens in clinical samples, and rpoB sequencing provides an additional identification tool for other species in the genus Vibrio.

Bacterial Typing Techniques↗
PubMed2006

A Trypanosoma cruzi antigen signals CD11b+ cells to secrete cytokines that promote polyclonal B cell proliferation and differentiation into antibody-secreting cells.

Microbial-induced polyclonal activation of B cells is a common event in several forms of infections, and is believed to play a crucial role both for enhancing the production of specific antibodies and for maintenance of B cell memory. Therefore, a major challenge in biomedical research is the identification of pathogen-derived products capable of rapidly mounting B cell expansion and differentiation. Here we report that glutamate dehydrogenase (GDH) stimulates polyclonal proliferation and differentiation of naive B cells. This stimulation was found to be T cell independent, but to absolutely require CD11b(+) cells. Moreover, we demonstrate that stimulation of CD11b(+) cells by GDH leads to the production of IL-6, IL-10 and B cell-activating factor (BAFF), all of which combine to powerfully induce B cell expansion. Importantly, IL-6 and IL-10 further drive B cell terminal differentiation into plasma cells by up-regulating critical transcription factors and immunoglobulin secretion. Our data provide the first evidence that a protozoan antigen can induce BAFF production by accessory cells, which in concert with other cytokines trigger polyclonal B cell activation.

Animals↗
PubMed2004

Duplex, triplex and quadruplex PCR for the preimplantation genetic diagnosis (PGD) of cystic fibrosis (CF), an exhaustive approach.

Most of cystic fibrosis (CF) pre-implantation genetic diagnosis (PGD) cases described to date are limited to the detection of DeltaF508. Beside this predominant mutation, over 1000 mutations have been identified, rendering the development of a mutation-based PGD protocol impracticable. This is the reason why we, as well as the others, have developed PGD strategies on the basis of the identification of the pathogenic haplotype instead of the mutation(s). In a previous article, we reported the conditions for the co-amplification of two intragenic polymorphic markers and the F508 locus. Here we describe an improved protocol allowing the additional amplification of two new intragenic markers, intron 1 CA repeat (I1CA) and IVS17bTA. This new protocol should, theoretically, allow us to provide a diagnosis to all couples requiring PGD for CF. Using single lymphoblasts, we have tested four different PCR configurations, including one duplex, two triplexes and one quadruplex PCR. All of them gave results compatible with a clinical application. The number of single lymphoblasts tested in each series varied from 89 to 155. PCR efficiency ranged from 95.4 to 100%. A complete haplotype was achieved for 83.2 to 90.7% of the tested cells, with an allele drop out (ADO) rate comprised between 6.0 and 11.6%. We present here three cases that we performed either with the former test (one case using the triplex PCR combining F508, IVS8CA and IVS17bCA) or with the new one (one case using the triplex combining F508, I1CA and IVS17bTA and one case using a quadruplex test). We obtained two single pregnancies.

Adult↗
PubMedDate not supplied

Molecular diagnosis of influenza.

The past decade has seen tremendous developments in molecular diagnostic techniques. In particular, the development of PCR technology has enabled rapid and sensitive viral diagnostic tests to influence patient management. Molecular methods used directly on clinical material have an important role to play in the diagnosis and surveillance of influenza viruses. Molecular diagnostic tests that allow timely and accurate detection of influenza are already implemented in many laboratories. The combination of automated purification of nucleic acids with real-time PCR should enable even more rapid identification of viral pathogens such as influenza viruses in clinical material. The recent development of DNA microarrays to identify either multiple gene targets from a single pathogen, or multiple pathogens in a single sample has the capacity to transform influenza diagnosis. While molecular methods will not replace cell culture for the provision of virus isolates for antigenic characterisation, they remain invaluable in assisting our understanding of the epidemiology of influenza viruses.

Adolescent↗
PubMed1985

The epidemiology of diabetes in Swedish children 0-14 years--a six-year prospective study.

Since 1 July 1977, all newly diagnosed diabetic children in Sweden aged 0-14 years have been reported to a central register. During the first 6 years, 2300 newly diagnosed diabetic children out of a population of 1.6 million children were registered. The degree of certainty was close to 100%. The mean of the yearly incidence rate for the whole 6 year period was 23.6 per 100000. The prevalence of insulin dependent diabetes mellitus on 1 July 1980 was 1.48 per 1000 and 1.52 on 1 July 1983. Comparing the first and second 3-year periods, an increase was found (22.7-25.1 per 100000). This increase was consistent when analyzing incidence rates by age, sex, and geographical distribution. Cumulative incidence rates revealed a risk of developing diabetes by the age of 15 years of 3.6 per thousand for boys and 3.2 per thousand for girls. The higher incidence for boys was consistent throughout the study period. Seasonal variations in the incidence rate were also consistent, showing yearly incidence peaks in the autumn and winter months. Incidence peaks were noted for both sexes in the pubertal ages. Age- and sex-standardized morbidity ratios varied significantly within the country. 12.8% of the probands had a first degree relative with Type 1 diabetes, and it was twice as common that this relative was a father as a mother. The high and rapidly increasing incidence of Type 1 diabetes in a genetically stable population such as Sweden calls for case-control studies directed towards the identification of environmental pathogens.

Adolescent↗
PubMed1983

Autofluorescence emission spectra of neuronal lipopigment in a case of adult-onset ceroidosis (Kufs' disease).

The study reports characteristics of the autofluorescence emission spectra from abnormal accumulations of intraneuronal lipopigment in a case of adult-onset neuronal ceroidosis (Kufs' disease). These are compared with emission spectra from 14 non-diseased brains, and from cases of childhood-onset neuronal ceroidoses (Batten's disease) and animal ceroidoses which have been previously reported. The emission spectra derived from the case of Kufs' disease could be distinguished from the spectra from the non-diseased brains and from the cases of Batten's disease and animal ceroidoses. The characteristics of an emission spectrum probably reflect the composition of the lipopigment, and spectral analysis may aid the identification of different pathogenic mechanisms which underly the various types of neuronal ceroidosis.

Adult↗
PubMedDate not supplied

Penetration of ofloxacin into nasal tissues.

The average concentration in 75 female/male patients was 1.90 +/- 1.10/1.55 +/- 0.62 mg/l in serum, 0.81 +/- 0.42/0.74 +/- 0.48 mg/kg in bone, 2.40 +/- 1.16/1.94 +/- 0.87 mg/kg in cartilage, and 2.35 +/- 1.28/1.99 +/- 1.02 mg/kg in mucosa. In each case the highest serum or tissue concentrations were observed 2 hours after the last ofloxacin administration (serum 2.60 +/- 0.41 mg/l, bone 1.10 +/- 0.75 mg/kg, cartilage 3.40 +/- 0.86 mg/kg, mucosa 3.50 +/- 1.05 mg/kg), but even 8 hours after the last dose the levels still exceeded the MIC 90% of e.g. Pseudomonas aeruginosa, Staphylococcus aureus or Haemophilus influenzae. The clinical investigations carried out show that ofloxacin represents a new oral therapeutic agent of significant value in otorhinolaryngology, particularly in the treatment of problematic infections. As the investigations show, the level in healthy tissue is definitely within the therapeutic region for a period of 8 hours. In ENT (nasal and paranasal) infections, ofloxacin should be given for defined indications, e.g. for oral therapy of problematic infections due to P. aeruginosa, and if possible after identification of the pathogen.

Adolescent↗
PubMed2006

Mammalian toll-like receptors: from endogenous ligands to tissue regeneration.

Following injury a complex but well-orchestrated cellular response stimulating wound healing and tissue regeneration is induced. The balance of different cytokines, growth factors and cells is important in regulating tissue reorganisation. The immune system is critically involved in this process. Toll-like receptors (TLRs) are essential to the innate immune system, recognising microbial pathogens. The recent identification of endogenous ligands of TLRs suggests that they function not only to induce defensive antimicrobial immune responses but also as a sensitive detection system to initiate tissue regeneration after injury. Here we present an overview of TLRs and their endogenous ligands, and also review the roles of TLRs in inducing tissue regeneration after injury and in maintaining homeostasis. The identification of endogenous TLR ligands and their involvement in inducing tissue regeneration will provide new options to improve tissue reorganization after injury.

Animals↗
PubMed2006

[Mycological laboratory].

The clinical presentation of fungal infections of the skin, hair, or nails may be varied and confusing. To avoid delays in treatment and early spread of the infection, adequate diagnostics are necessary. This requires both the clinical expertise of the physicians as well as the capability to perform various laboratory tests. Classical methods are based on microscopy, different culture procedures, and histological examination. Genotypic approaches for the detection and identification of fungal pathogens have improved the speed and accuracy of laboratory diagnosis in the last few years, although further evaluation using molecular biology-based methods should be routinely applied in practice.

Antifungal Agents↗
PubMed2003

Haemophilus parainfluenzae: an underdiagnosed pathogen of biliary tract infections?

Presented here is a case of monobacterial peritonitis complicating cholecystitis and caused by an uncommon agent of gastrointestinal infections, Haemophilus parainfluenzae. The pathogenic role of this organism in digestive infections, particularly in those of the biliary tract, has been reported increasingly though sporadically. Indeed, it has been shown to be a coloniser of the gastrointestinal tract, and a recent hypothesis of an ascending route of infection to the biliary tract has been postulated to partly explain its pathogenicity. More frequent identification of Haemophilus parainfluenzae as a causal agent of biliary tract infection would probably be obtained through the use of specific culture media, since its potential implication has been demonstrated.

Anti-Bacterial Agents↗
PubMed1993

Invasive aspergillosis in patients with acquired immunodeficiency syndrome: report of 33 cases. French Cooperative Study Group on Aspergillosis in AIDS.

PURPOSE: Acquired immunodeficiency syndrome (AIDS)-associated invasive aspergillosis (IA) is a rare condition, which is mainly reported as isolated cases either antemortem or at autopsy. The role of AIDS itself is controversial, because many of the reported patients exhibited the classic risk factors such as neutropenia and steroid therapy. The aims of this study were to report 33 patients with IA during AIDS and their outcome, focusing on the risk factors and the value of diagnostic procedures. PATIENTS AND METHODS: Thirty-three patients from 17 different medical centers in France were retrospectively included in the study. For pulmonary IA, we defined two types of patients: those with "confirmed IA," describing all the patients with histologically proven disease, and those with "probable IA," who had the development of a new pulmonary infiltrate on chest radiograph and a positive bronchoalveolar lavage (BAL) fluid culture for Aspergillus species without identification of other pathogens. For extrapulmonary IA, the diagnostic criteria included both positive histology and culture. RESULTS: Of the 33 cases included in this series, 91% were recorded during the last 3 years (1989 to 1991), suggesting that aspergillosis is an emerging complication in AIDS. Approximately 50% of the patients did not exhibit any classic risk factor, i.e., neutropenia and steroid treatment; almost all patients had a CD4 cell count less than 50/mm3. The mycologic culture from BAL was the method of choice for the diagnosis of invasive pulmonary disease because it was known to correlate well with histologic findings obtained either antemortem or postmortem. Of 28 patients with a positive BAL culture for Aspergillus, 15 underwent a biopsy or autopsy and 14 were positive at histology. Serum antigen detection was positive in only 4 of 16 tested patients. Clinical and radiologic signs did not differ from those observed in neutropenic patients without human immunodeficiency virus, except for the higher incidence of neurologic complications in AIDS. Interestingly, we observed three cases of invasive necrotizing tracheobronchial aspergillosis with acute dyspnea and wheezing. The use of amphotericin B (0.5 mg/kg/d) and/or itraconazole (200 to 600 mg/d) was most often unsuccessful. Only four patients experienced clinical and radiologic improvement. The mean interval between the diagnosis of IA and death was 8 weeks (range: 3 days to 13 months). CONCLUSIONS: This study suggests that aspergillosis is an important life-threatening condition in the advanced stage of AIDS. It requires an early diagnosis with BAL fluid culture and careful therapeutic evaluation.

AIDS-Related Opportunistic Infections↗
PubMed1994

Microbiologic factors in endodontology.

The role of microorganisms in the cause of endodontic lesions has been intensively investigated. Bacterial components such as endotoxin and other cell wall components are implicated in the development of pulpal and periapical inflammation. Newer anaerobic microbiologic techniques have facilitated accurate and reproducible identification of endodontic pathogens, some of which have been reclassified. This article reviews and correlates newer microbiologic findings with clinical symptoms.

Bacteria, Anaerobic↗