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PubMed2005

Etiologies of nongonococcal urethritis: bacteria, viruses, and the association with orogenital exposure.

BACKGROUND: The purpose of the present study was to determine pathogens and behaviors associated with nongonococcal urethritis (NGU) and the usefulness of the urethral smear in predicting the presence of pathogens. METHODS: We conducted a case-control study of men with and without symptoms of NGU. Sexual practices were measured by questionnaire. First-stream urine was tested for Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma parvum, U. urealyticum, herpes simplex virus (HSV)-1, HSV-2, adenoviruses, and Gardnerella vaginalis by polymerase chain reaction. RESULTS: C. trachomatis (20%), M. genitalium (9%), adenoviruses (4%), and HSV-1 (2%) were more common in cases with NGU (n = 329) after age and sexual risk were adjusted for (P< or =.01); U. urealyticum, U. parvum, and G. vaginalis were not. Infection with adenoviruses or HSV-1 was associated with distinct clinical features, oral sex, and male partners, whereas infection with M. genitalium or C. trachomatis was associated with unprotected vaginal sex. Oral sex was associated with NGU in which no pathogen was detected (P < or = .001). Fewer than 5 polymorphonuclear leukocytes (PMNLs) per high-power field (HPF) on urethral smear were present in 32%, 37%, 38%, and 44% of cases with C. trachomatis, M. genitalium, adenoviruses, and HSV, respectively. CONCLUSION: We identified adenoviruses and HSV-1 as significant causes of NGU with distinct clinical and behavioral characteristics and highlighted the association between insertive oral sex and NGU. A urethral PMNL count of > or =5 PMNLs/HPF is not sufficiently sensitive to exclude pathogens in men with urethral symptoms.

Adenovirus Infections, Human↗
PubMed1989

[Factors on delayed recovery of otitis media with effusion in children--clinical and bacteriological study].

The resolution of middle ear effusions (MEE) of children with otitis media with effusion (OME) who underwent myringotomy for the bacteriological examination was analyzed in terms of the culture results and the clinical features. The present study consisted of 193 children (258 ears), and the MEE from 77 ears (30%) were culture positive and the respiratory pathogens were detected from 44 ears (17%). Each child was then assigned to receive either a more than two-week course of antibiotics, cefaclor (CCL) or not. At one month following entry, 53 (55%) out of 97 ears in CCL-treated group were effusion-free compared with 31 (40%) out of 78 ears in the control group (P less than 0.05). In the control group, the resolution of MEE was significantly poor in the recurrent cases and the cases with pathogen positive-MEE. The presence of accompanying diseases such as adenoid vegetation, chronic sinusitis and allergy, however, was not related to the resolution of MEE. On the other hand, the cure rate of the cases with pathogen positive-MEE and recurrent cases in the CCL-treated group showed significant improvement. Furthermore, the cases accompanying adenoid vegetation and chronic sinusitis tended to become effusion-free after the antibiotic treatment. Therefore, the persistent bacterial infection in the middle ear and/or surrounding organs such as adenoid plays possibly an important role in the delayed recovery of OME. Antibiotics treatment could increase, to some extent, the resolution of MEE in cases with OME.

Adolescent↗
PubMed2000

Inhibitory effects of collagen on the PCR for detection of Clostridium perfringens.

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.

Clostridium perfringens↗
PubMed1996

Early recognition of Streptococcus pneumoniae in patients with community-acquired pneumonia.

The objective of this study was to assess the predictive value of signs, symptoms, and rapidly available laboratory parameters for pneumococci in community-acquired pneumonia (CAP). A prospective study on patients with CAP who were admitted to hospital was conducted. Clinical and laboratory data were collected according to a protocol. Two hundred sixty-eight patients aged 18 years or older, not living in a nursing home or not admitted to hospital within one week of this admission, with a new infiltrate on the chest radiograph consistent with pneumonia were included. According to microbiological and serological tests, patients were allocated to one of two aetiological groups, Streptococcus pneumoniae or "other pathogens". Seventy-three variables were examined for a correlation with one of the aetiological categories by means of univariate and multivariate analysis. The resulting discriminant function was considered a clinical test for which posttest probabilities for pneumococcal pneumonia were calculated. Streptococcus pneumoniae was demonstrated in 79 patients and other pathogens in 83; no pathogens were detectable in 106 patients. The variables "cardiovascular disease", "acute onset", "pleuritic pain", "gram-positive bacteria in the sputum Gram stain", and "leucocyte count" correctly predicted the cause of CAP in 80% of all cases in both groups. Depending on the prevalence of Streptococcus pneumoniae, posttest probabilities for pneumococcal pneumonia were up to 90%. It is concluded that data on history, together with the result of the Gram stain of sputum and the leucocyte count, can help to distinguish Streptococcus pneumoniae from other pathogens causing CAP.

Acute Disease↗
PubMed1996

Specific detection of Candida albicans and Candida tropicalis by fluorescent in situ hybridization with an 18S rRNA-targeted oligonucleotide probe.

In situ hybridization of whole cells with rRNA-targeted, fluorescently labelled oligonucleotide probes is a powerful method to specifically detect microorganisms in their natural habitat without cultivation and subsequent identification by phenotypic characterization. To examine the use of this method for the specific detection of pathogenic Candida species, we have designed an oligonucleotide probe which binds to the 18S rRNA of C. albicans and C. tropicalis, the two most important pathogenic Candida species, and differentiates them from other clinically relevant species. After establishing suitable hybridization conditions, we confirmed the specificity of our probe O20 in RNA dot blot hybridizations with a series of reference strains and clinical isolates of medically important Candida species. All C. albicans and C. tropicalis strains hybridized with the probe, whereas all strains of C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. kefyr and C. lusitaniae did not. When we used the fluorescently labelled probe O20 to specifically detect single cells of the two target species by in situ hybridization, both C. albicans and C. tropicalis reacted strongly with the probe and could be clearly differentiated from C. krusei and C. parapsilosis, although the latter organism contains only two nucleotide mismatches in the probe target region. This discrimination capacity was also seen when mixed suspensions of C. albicans and C. parapsilosis were hybridized with the probe. After infection of a human endothelial cell line with C. albicans and C. krusei, C. albicans cells adhering to the endothelial cells were easily distinguishable from the C. krusei cells by fluorescent in situ hybridization with probe O20. In addition, germ tubes and hyphae of C. albicans were also efficiently labelled. The application of fluorescently labelled rRNA-targeted oligonucleotide probes therefore appears to be a valuable tool for the specific detection and identification of different members of the genus Candida, which does not require any cultivation.

Candida↗
PubMed2006

Vaccines developed for H5 highly pathogenic avian influenza in China.

Since the first detection of highly pathogenic H5N1 avian influenza virus from sick goose in Guangdong province in China in 1996, scientists in China started to develop vaccines for avian influenza pandemic preparedness. An H5N2 inactivated vaccine was produced from a low pathogenic virus, A/turkey/England/N-28/73, and was used for the buffer zone vaccination in the H5N1 outbreaks in 2004 in China. We also generated a low pathogenic H5N1 reassortant virus A/Harbin/Re-1/2003 (Re-1) that derives its HA and NA genes from GSGD/96 virus and six internal genes from the high-growth A/Puerto Rico/8/34 (PR8) virus by using plasmid-based reverse genetics. The inactivated vaccine derived from Re-1 strain could induce more than 10 months protective immune response in chickens after one dose inoculation, and most importantly, this vaccine is immunogenic for geese and ducks. An H5N1 fowlpox vectored live vaccine was also generated by inserting the HA and NA genes of GSGD/96 virus in the genome of a fowlpox vaccine strain. Laboratory tests indicated that after one dose of immunization of this vaccine, chickens could develop an over than 40 weeks protective immune response against H5N1 virus challenge.

Animals↗
PubMedDate not supplied

Quantification of PCR products using microparticles and flow cytometry.

The analysis of genetic biomarkers has become an important tool in clinical diagnostics. This includes the identification of disease-related genetic alterations, the detection of pathogenic infective germs on DNA or RNA level and the quantification of the expression of marker genes indicating an altered physiological status. It has been previously described that the combination of polymerase chain reaction (PCR), microparticles and flow cytometry represents a universal platform technology for the routine analysis of such biomarkers. Here we demonstrate the applicability and flexibility of this technology by means of various applications. The quantification of interferon gamma (IFNG) mRNA in irradiated white blood cells is shown as well as the detection of latent infections with cytomegalovirus (CMV). Besides the quantification of single amplification products, the flow cytometric assay is also capable of analysing products of a multiplex PCR. As an example, we describe the identification of spontaneous deletions in the genome of a hybrid cell line using a co-amplified gene (RAB1) essential for the cell survival as an internal control. Furthermore, we show that the use of a green laser (532 nm, 50 mW) substantially increased the sensitivity of the assay compared to conventional flow cytometers using a 488 nm (25 mW) laser. We conclude that the analysis of PCR products using microparticles and flow cytometry fulfils the criteria of clinical routine diagnostics regarding (i) sensitivity, (ii) specificity, (iii) reproducibility and (iv) automatibility.

Animals↗
PubMed2000

A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR.

Rapid nucleic acid-based methods to detect human pathogens in foods are dependent on the reliability of the DNA or RNA extraction method used. Skim milk, non-fat dry milk, Cheddar and Brie cheese, and reconstituted whey powder were seeded with serially diluted (10(0)-10(7) cfu 10 ml(-1)) Escherichia coli O157:H7 and subjected to DNA extraction (i) directly from the food product using a solvent-based procedure and (ii) using a guanidinium isothiocyanate (GITC) procedure after previous bacterial concentration. Both the efficiency of DNA extraction and the overall PCR detection limits were evaluated. In almost all instances, the total DNA yield using the solvent method was greater than that obtained for the concentration method. However, the purity of the DNA obtained after bacterial concentration was significantly better than that obtained after organic extraction alone. PCR detection limits after each DNA recovery method varied with the specific food, ranging from 10(1) to 10(4) cfu ml(-1) for all products except whey powder. DNA yields and subsequent PCR detection limits for reconstituted whey powder were extremely poor, and neither procedural changes nor the addition of PCR enhancement agents were able to improve recovery and/or detection. It is concluded that the efficiency of DNA extraction is an extremely important and frequently overlooked variable impacting the overall detection limits of PCR-based detection strategies.

Animals↗
PubMed2004

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection.

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Animals↗
PubMed1993

Rapid diagnostic tests for infectious ocular disease.

There is a current and ongoing need for the rapid, accurate diagnosis of ocular infection and the prompt initiation of appropriate therapy to prevent visual loss, maximize use of health care resources, and reduce physicians' medicolegal liability. For the busy clinician, and ideal rapid diagnostic test to determine ocular pathogens would be one whose result is available before the patient leaves the doctor's office, so that optimal treatment can be promptly instituted. Today, the major rapid diagnostic tests available to the busy clinician are the clinical examination and the stained microscopical smear of conjunctival and corneal specimens. The latter technique is grossly underutilized and would contribute significantly to improved accuracy in early diagnosis of ocular infections. In the near future, improved enzyme immunoassays and simplified PCR kits will provide for highly sensitive and specific diagnostic capabilities for detecting ocular pathogens in the laboratory and, possibly, office settings.

Clinical Laboratory Techniques↗
PubMed2002

Role of infections in the manifestation or reactivation of inflammatory bowel diseases.

The etiology of inflammatory bowel disease (IBD) is unknown. In addition to genetic and environmental factors, microorganisms have been discussed as possibly playing an important role. Recent reports in the literature do not suggest that a specific persistent infection causes IBD, but indicate that enteric pathogens could cause initial onset of IBD and are associated with reactivation of quiescent disease. Despite their self-limited character, these infections initiate a cascade of inflammatory events leading to chronic, relapsing disease in a genetically susceptible host ("hit-and-run" hypothesis). Epidemiological and microbiologic studies suggest that enteropathogenic microorganisms play a substantial role in the clinical initiation and relapses of IBD. However, similar to traveler's diarrhea, the frequency of infections in the first manifestation and in relapses of IBD is probably understated, due to the problems in detecting enteric pathogens. Thus microbiologic screening is helpful in patients with flares of IBD for optimal medical treatment.

Bacterial Infections↗
PubMed1993

Detection of seven species of pathogenic leptospires by PCR using two sets of primers.

Two sets of primers derived from genomic DNA libraries of Leptospira serovars icterohaemorrhagiae (strain RGA) and bim (strain 1051) enabled the amplification by PCR of target DNA fragments from leptospiral reference strains belonging to all presently described pathogenic Leptospira species. The icterohaemorrhagiae-derived primers (G1/G2) enabled amplification of DNA from L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. santarosai and L. meyeri, whereas the bim-derived primers (B64-I/B64-II) enabled the amplification of L. kirschneri. Southern blot and DNA sequence analysis revealed inter-species DNA polymorphism within the region spanned by primers G1 and G2 between L. interrogans and various other Leptospira species. Using a mixture of primer sets G1/G2 and B64-I/B64-II, leptospires of serovars icterohaemorrhagiae, copenhageni, hardjo, pomona, grippotyphosa and bim were detected in serum samples collected from patients during the first 10 days after the onset of illness.

Base Sequence↗
PubMed2026

Genome-wide characterization of NOD-like receptor genes links NLR repertoire evolution to spleen immune responses after Aeromonas hydrophila challenge in the Chinese spiny frog (Quasipaa spinosa).

NOD-like receptors (NLRs) are cytosolic pattern-recognition receptors that detect pathogen-associated and damage-associated molecular patterns and mediate innate immune signaling in vertebrates. However, the genomic repertoire, evolutionary diversification, and infection-associated expression of NLR genes remain poorly defined in non-model amphibians. In this study, 66 NLR genes were identified from the Chinese spiny frog (Quasipaa spinosa) genome and designated as QsNLR1-QsNLR66. These genes were unevenly distributed across chromosomes and were classified into three phylogenetic groups, with most members exhibiting conserved motif architectures. Gene duplication analysis indicated that dispersed duplication was the main contributor to QsNLR expansion. Synteny analysis detected five conserved orthologous gene pairs between Q. spinosa and Pelophylax nigromaculatus, suggesting partial conservation of NLR genomic organization between the two amphibians. Ka/Ks analysis showed that several duplicated gene pairs, including NLRC3-like/QsNLR36 and NLRC3-like/QsNLR50, exhibited Ka/Ks ratios greater than one, suggesting potential sequence divergence after duplication. Spleen RNA sequencing (RNA-seq) after Aeromonas hydrophila challenge revealed enrichment of immune-related Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Weighted gene co-expression network analysis linked several QsNLRs to infection-associated modules, among which QsNLR57 was co-expressed with CYBB, ADAM17, SPI1, and HK2. RT-qPCR using time-matched phosphate-buffered saline (PBS) controls showed distinct temporal patterns, with stronger induction of QsNLR29, QsNLR57, and QsNLR66 and weaker or delayed responses of QsNLR50 and QsNLR56. These results characterize the NLR repertoire of Q. spinosa and identify infection-associated QsNLR candidates for future studies of antibacterial immunity in amphibians.

Animals↗
PubMed2001

DNA array technology and diagnostic microbiology.

Near instantaneous detection of pathogens from clinical material, combined with simultaneous prediction of their antimicrobial resistance profiles, would revolutionize the impact of microbiology on the management of infection. Array-based assays allow a range of characteristics to be rapidly and simultaneously determined. At present these systems have found their primary role as research tools for the monitoring of mRNA expression in the form of DNA microarrays or 'chips'. As fabrication costs reduce and validated targeted arrays are developed, it is inevitable they will be used for more routine applications. Microfluidics offers the exciting possibility of combining purification, amplification and detection in a single disposable device; microarrays are particularly suitable for use within these systems. Arrays will become an important tool for clinical diagnostics.

Animals↗
PubMed1991

Relation of counts of microbial species to clinical status at the sampled site.

The purpose of the present investigation was to relate clinical characteristics at a site to the frequency of detection, absolute counts and proportions of 14 subgingival species. Subgingival plaque samples were removed by curette from the mesial surface of 2299 teeth in 3 healthy and 87 subjects with periodontal attachment loss. Samples were dispersed, diluted and plated on Trypticase soy agar supplemented with 5% sheep blood. After 7 days of anaerobic incubation, colonies were lifted onto nylon filters, lysed and the DNA fixed to the filters. Digoxygenin-labeled DNA probes were used to identify colonies of each test species. Measurements of pocket depth, attachment level, recession, redness, bleeding on probing and suppuration were made at each sampled site. Total viable counts at sites ranged from 10(3) to greater than 10(8) and were strongly related to pocket depth. Mean total counts at sites less than 3 mm averaged 4.6 x 10(6), while mean counts at sites greater than 7 mm averaged 2.0 x 10(7). Species enumerated and % of sites colonized were as follows; V. parvula 44; S. sanguis II 36; B. intermedius I 33; C. ochracea 31; B. intermedius II 30; S. sanguis I 29; B. gingivalis 27; S. intermedius 25; P. micros 24; W. recta 23; F. nucleatum ss vincentii 18; B. forsythus 15; A. actinomycetemcomitans serotype a 10; A. actinomycetemcomitans serotype b 8. Counts of B. intermedius II were higher at sites which exhibited gingival redness while B. intermedius I was higher at sites which bled on probing. A. actinomycetemcomitans serotype b was more frequent and at higher mean % at sites without recession. The opposite was true for S. sanguis II. B. gingivalis was somewhat more prevalent and at higher levels at suppurating sites. B. gingivalis, B. intermedius I and II and B. forsythus were found more frequently and at higher levels at sites with deeper pockets, while V. parvula was less prevalent at sites with pocket depths less than 4 mm. B. gingivalis, B. intermedius I and A. actinomycetemcomitans serotype b increased with increasing pocket depth in both localized and widespread disease subjects, but mean counts were higher in the localized disease subjects at any pocket depth. Only W. recta was found at higher levels at deep sites in widespread disease subjects when compared with similar sites in localized disease subjects. No suspected pathogens were detected in 38% of shallow sites, 31% of intermediate sites and 22% of deep sites, 2/3 of deep pockets, but less than 1/2 of shallow pockets harbored at least 2 of the suspected pathogens.

Aggregatibacter actinomycetemcomitans↗
PubMed2000

Detection of multiple potato viruses using an oligo(dT) as a common cDNA primer in multiplex RT-PCR.

A novel usage of multiplex reverse transcription polymerase chain reaction (m-RT-PCR) for simultaneous detection of multiple viruses is reported. By use of an oligo(dT), as a common primer, nearly full-length cDNAs can be synthesized. Furthermore, combining an oligo(dT) primer with a specific antisense primer can be used to simultaneously prime reverse transcription of both polyadenylated and non-polyadenylated RNAs. Four viral genera including five potato viruses [(carlavirus (PVS), polerovirus (PLRV), potexvirus (PVX), potyvirus (PVA and PVY))] and a viroid genus including a viroid genome (pospiviroid (PSTVd)) were used to develop various formats of m-RT-PCR. In artificially created viral RNA mixtures, all six RNA pathogens were detected successfully by uniplex- and m-RT-PCR. In naturally infected field grown tubers, m-RT-PCR detected infection of two to three viruses, which were present in the tubers.

DNA, Complementary↗
PubMed1994

Characterization of turbot (Scophthalmus maximus) associated bacteria with inhibitory effects against the fish pathogen Vibrio anguillarum.

More than 400 isolates from the intestine and the external surface of farmed turbot, as well as from fish food and hatchery water were screened for inhibitory effects against the fish pathogen Vibrio anguillarum HI 11345 and seven other fish pathogens. The bacteria with inhibitory effects were then characterized with regards to their sites of colonization, especially the intestinal regions and sites within each region. No correlations between the different biochemical phena, site of colonization and inhibitory effect could be found. The potential of seven of these strains for adhesion to intestinal mucus from turbot was studied. Rapid growth of V. anguillarum in intestinal mucus was measured, hence it is feasible that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for in vitro adhesion to and growth in fish intestinal mucus than did the pathogen and skin mucus isolates. Two of the strains isolated from the intestine were studied for their inhibitory kinetics and one strain for the potential of in vivo colonization. The molecular weight of the inhibitory component was below 1000 dalton in 65% of the strains isolated. For the other 35% the inhibitory component ranged between 1000 D-6000 D in dialysis cut off experiments. One week after oral administration, one such isolate still accounted for 45% of the total c.f.u. in the intestinal mucus of 5 g turbot. In preliminary experiments we demonstrated that it is possible to detect the pathogen in intestinal mucus with a fluorescently labelled oligonucleotide probe directed against a specific part of the intracellular rRNA of V. anguillarum.

Animals↗