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[The outlook for an improvement in the agents for detection based on immunofluorescence analysis].

The review deals with prospects for improving the agents for rapid indication of biological agents by a combination of currently available immunochemical and molecular genetic analyses and the latest developments in the design of luminescent tracers, techniques of recording superweak luminous fluxes in the photon count mode and laser spectroscopies. Immunosensor technologies and homogeneous immunoassay are shown to be optimal for the studies to rapidly detect pathogens and toxins. It is most advisable to employ highly sensitive, specific, and efficient methods of molecular hybridization and multicomponent solid-phase immunoassay by recording biospecific binding products in the mode of time luminescence resolution for environmental studies and clinical laboratory diagnoses in resident laboratories. Continuous water and air follow-up monitoring involves the analysis by using immunosorbents, as well as immunosensors and flow cell fluorimetry is noted to become an important independent line in the development of biological indication agents under the increasing man-made influence of the environment.

Animals↗

Fatal herpesvirus-6 encephalitis in a recipient of a T-cell-depleted peripheral blood stem cell transplant from a 3-loci mismatched related donor.

Human herpesvirus-6 (HHV-6), like all the other herpes viruses, remains latent in host cells after primary infection but can be reactivated in immunocompromised patients causing fever, skin rash, bone marrow (BM) suppression, pneumonitis, sinusitis and meningoencephalitis. We describe the case of a man with chronic myelogenous leukemia who developed encephalitis associated with acute graft-versus-host disease two months after a T-cell-depleted mismatched peripheral blood stem cell transplant. Magnetic resonance images of the brain revealed multiple bilateral foci of signal abnormality. HHV-6 was the only pathogen detected in cerebrospinal fluid by PCR. Treatment with both ganciclovir and foscarnet was unsuccessful and the patient gradually deteriorated and died. Other cases of HHV-6 encephalitis after bone marrow transplantation are reviewed.

Cytomegalovirus Infections↗

[Use of polymerase chain reaction for pediatric diagnosis].

Basic principles of main molecular biology techniques: polymerase chain reaction (PCR) and its variants; asymmetric PCR, ARMS, nested PCR, multiplex PCR and competitive PCR are discussed. Nucleic acids techniques are making increasing progress in molecular diagnostics. Sequence analysis of amplified DNA allows better identification of the pathogen, detection of mutant genes and more accurate prognosis and therapy of certain diseases.

Child↗

Environmental air pollution in an intensive care unit for nephrology and dialysis.

The quality of indoor air depends on external pollutant concentrations and on internal sources, such as heating and air conditioning systems, building materials, ventilation, cleaning products, personnel and their activity. This study assessed environmental air pollution in an intensive care unit (ICU) for nephrology and dialysis. Air-dispersed particulate pollution was measured using a gravimetric method and spectroscopic photocorrelation. Microbiological pollution was evaluated by passive and active collection. Particulate concentrations exceeded recommended limits in some of the environments. There was a prevalence of small particulates, which are the most harmful type of all. An overall evaluation of bacterial pollution showed low levels of contamination in some of the rooms. In none of the environments we were able to detect pathogens such as Aspergillus fumigatus, methycillin-resistant Staphylococci or toxin-producing fungi.

Air Microbiology↗

[A comparison of polymerase chain reaction with other two widely used methods in detection of Porphyromonas gingivalis].

OBJECTIVE: To compare the efficiency of polymerase chain reaction (PCR) with cultural method and indirect immunofluorescence (IF) in detection of Porphyromonas gingivalis (P.g) in oral plaque samples. METHODS: 92 subgingival plaque samples were collected from patients of three kinds of periodontal disease. A program using PCR to detect Fimbrilin gene (Fim A) was designed to detect P.g. The efficiency of this PCR method was compared with cultural method and IF. RESULTS: The positive-rate of PCR was 94.6%, while that of cultural method and IF was only 48.9% and 68.5% respectively. The difference was significant in statistical analysis (P < 0.001). CONCLUSION: PCR is more sensitive than culture method and IF in detecting pathogens of periodontal diseases. The study provides a simplified and efficient method for oral clinical use.

Adult↗

Bacterial etiology of otitis media with effusion; focusing on the high positivity of Alloiococcus otitidis.

The etiology of otitis media with effusion (OME) is unclear. The bacterial analyses of middle ear effusion (MEE) in OME may reveal important information regarding its etiology. Alloiococcus otitidis, Heamophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were investigated by using microbiologic culture and a multiplex PCR method in the middle ear fluid of 32 children (54 samples) with chronic OME. PCR yielded positive results in 18 (33.3%) middle ear effusions while culture resulted positive for 3 (5.6%). The PCR method detected A. otitidis in 10 (18.5%) specimens, H. influenzae in 7 (13%), M. catarrhalis in 4 (7.4%) and S. pneumoniae in 2 (3.7%) specimens. The multiplex PCR method enhances the detection rate significantly compared to that of the conventional culture method. A. otitidis is the most common detected pathogen in the MEE of the OME.

Adolescent↗

[Detection of Toxoplasma gondii DNA in human lymph node tissue by in situ hybridization].

AIM: To detect the presence of Toxoplasma gondii in lymph node tissue in patients with Toxoplasma infection. METHODS: T. gondii (RH strain) specific DNA fragment clones were obtained by using PCR and gene recombination technique. The DNA fragments used as hybridization probes were labelled with digoxigenin by random primer method. The technique of in situ hybridization (ISH) was used to detect T. g DNA in the lymph node sections. RESULTS: Four out of 120 samples T. g DNA were found positive, one with Hodgkin's disease (HD) (1/32), one with non-Hodgkin's lymphoma (NHL) (1/41) and 2 with chronic lymphadenitis (CL) (2/47). The total positive rate was 3.3%. It was demonstrated that this highly specific probe could detect 10 pg of the total RH strain T. g DNA. CONCLUSION: ISH was applicable in detecting pathogens in the lymph node tissues of individuals with Toxoplasma infection.

Animals↗

[Prevention of intramammary infections in dairy cows by the use of a premilking teat dip method with a foaming iodophor dip agent].

In this study we investigated the efficacy of premilking teat dipping with a foaming iodophor teat dip in a negative controlled field study. Incidence of new intramammary infections (IMI), incidence of clinical mastitis, influence on somatic cell count (SCC) and the characteristics of udder tissue and teats were used as parameters to evaluate clinical efficacy. Predipping was compared with a negative control using a split-udder experimental design. Right teats were predipped with a foaming disinfectant containing 0.27% iodine while left teats served as controls. The latter were conventionally cleaned with damp cloth towels and dried manually with disposable paper towels ("best cleaning practice"). All teats were dipped after milking with the same dip. There were no differences between treated and control quarters with respect to incidence of new IMI during the study period (treated quarters: 6.6% vs. untreated: 6.95%), incidence of clinical mastitis (30 cases in the treatment group vs. 39 cases in the control group) and geometric mean of SCC of quarter milk samples. Spectrum of detected pathogens was also comparable. Condition of udder tissue and teat ducts did not differ between treated and control quarters.

Administration, Topical↗

Establishment and analysis of specific DNA patterns in 16S-23S rRNA gene spacer regions for differentiating different bacteria.

OBJECTIVE: To establish the specific 16S-23S rRNA gene spacer regions in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis. METHODS: A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA from sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 26 species was amplified by PCR, and further analyzed by RFLP, DNA cloning and sequences analysis. Furthermore, all specimens were examined by bacterial culturing and PCR-RFLP analysis. The evaluation of these assays in practical clinic practice was also discussed. RESULTS: Restriction enzyme analysis revealed one, two or three bands or more observed among the 26 different standard strains. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction with human genomic DNA, fungus or virus. Fourteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. The only difference between K.pneumoniae and E. durans was located at the site of the 779th nucleotide according to the sequence analysis and only XmaIII digestion could distinguish one from another. Of 42 specimens from septicemic neonates, 15 were identified as positive by blood culture at a rate of 35.7%. However, 27 specimens identified as positive by PCR, with a rate of 64.2%, a method significantly more effective than blood culture (P < 0.01). Of 6 cerebrospinal fluid (CSF) specimens, one tested positive for S.epidermidis was also positive by PCR, two culture negative were positive by PCR and diagnosed as S.epidermidis according to the DNA pattern. One positive for C.neoformans was negative by PCR. The other two specimens were negative by both PCR and culture. CONCLUSIONS: The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in providing a new technique for detecting pathogens in clinical bacterial infections.

Bacteria↗

Milkborne general outbreaks of infectious intestinal disease, England and Wales, 1992-2000.

From 1 January 1992 to 31 December 2000, 27 milkborne general outbreaks of infectious intestinal disease (IID) were reported to the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC). These outbreaks represented a fraction (2%) of all outbreaks of foodborne origin (N = 1774) reported to CDSC, but were characterized by significant morbidity. Unpasteurized milk (52%) was the most commonly reported vehicle of infection in milkborne outbreaks, with milk sold as pasteurized accounting for the majority of the rest (37%). Salmonellas (37%), Vero cytotoxin-producing Escherichia coli (VTEC) O157 (33%) and campylobacters (26%) were the most commonly detected pathogens, and most outbreaks were linked to farms (67%). This report highlights the importance of VTEC O157 as a milkborne pathogen and the continued role of unpasteurized milk in human disease.

Animals↗

[Epidemiological study on group infection of Angiostrongylus cantonensis in Changle City].

OBJECTIVE: To make etiological and epidemiological investigation on the infection of Angiostrongylus cantonensis in 8 pupils in Changle City. METHODS: 1. CSF of patients was examined with the conventional method to detect pathogens and eosinophiles. 2. The fecal samples of wild rodents were collected from the spot and examined microscopically to discover the first stage larvae of A. cantonensis. 3. Snails (Pila gigas) were collected in the spot. The smashed head tissue was examined for the third stage larvae of A. cantonensis. 4. The patient's clinical symptoms and physical signs were recorded with an emphasis on central nervous system. RESULTS: 1. Two larvae of the third stage of A. cantonensis were found in CSF of one patient. Eosinophiles occupied 68% of the cell number in average (ranged from 47% to 83%) in CSF of the 8 patients. 2. The infection rate of the first stage larvae of A. cantonensis was 39.3% (44/112) in feces of the rodents. 3. The infection rate of the third stage larvae of A. cantonensis was 40.0% (82/205) in the snails. 4. Major clinical manifestations in the 8 patients included: severe headache(8/8), dizziness(8/8), nausea(8/8), vomiting(8/8), lethargy(7/8), lower limb hypodynamia(7/8). CONCLUSION: The confirmation of severe infection of A. cantonensis in 8 child patients demonstrated that a natural nidus of angiostrongyliasis is present in Chengle City.

Adolescent↗

[Use of affinity interaction of biologically active substances in practice of public health system].

AIM: To determine the potentialities of use of affinity interaction of immobilized biologically active substances (bacterial cells or their fragments, toxins, antigens of various chemical nature, immunoglobulins, enzymes, gangliosides, etc.) for medical practice. MATERIALS AND METHODS: Emulsion polymerization of acrylamide monomers in the gaseous nitrogen current was used as a basic method for preparation of solid-phase magnetic immunosorbents (MIC). A procedure for preparation of siliceous MIC was also applied. The prepared MICs were used a solid phase in enzyme immunoassay and immunofluorescence assay and the recorded data were compared with those of studied conventionally used in practical medicine. RESULTS: The use of MIC made it possible to detect pathogens of particularly dangerous infections in large volumes of the samples contaminated with another microflora. With the proposed MIC, one can stand a good chance of surveying large contingents of the population, of obtaining the quantitative results in shorter periods to establish a diagnosis. With this, the sensitivity and specificity of immunoassays substantially increase. Whether MIC may be used as selective hemosorbents to remove specific antibodies from the blood of patients with rheumatic diseases for therapeutic purposes was studied. CONCLUSION: The findings are indicative of wide potentialities of use of affinity interaction of biologically active substances immobilized on inert carriers with the inserted magnetic material in the laboratory diagnosis of diseases of both infectious and autoimmune nature, which may be widely used in the in- and outpatient settings.

Antibodies↗

[Molecular diagnosis of the specific DNA patterns of 16S-23S rRNA gene of bacteria].

OBJECTIVE: To establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis. METHODS: A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis. RESULTS: The 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR. CONCLUSIONS: The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.

Bacterial Infections↗

[Study on Bartonella infection using molecular biological diagnostic techniques from China].

OBJECTIVE: To establish polymerase chain reaction (PCR) technique for the detection of specific genes related to species of genus Bartonella, and for diagnosing clinically suspected cat-scratch disease (CSD) case complicated with pneumonia on both lungs. The appearance of Bartonella infectious diseases calls for genus and species detection and tools for identification in order to make clinical diagnosis and carry on epidemiological studies. METHODS: One pair of primer TIle.455p-TAla.885n was designed based on the fact that tRNA(Ile)-tRNA(Ala) intergenic spacer region in 16S-23S rRNA intergenic spacer (ITS) of genus Bartonella were high variable sequences flanked by completely conserved tRNA-encoding genes. 16S-23S rRNA was longer than that which had been described in other bacteria. Two published pairs of primers were used to directly detect the specific gene fragments of Bartonella species DNA extracts from human blood, followed by PCR product Sequencing and nucleotide base sequence analysis. RESULTS: Amplification products of the three pairs of primers had the same predicted size of those in Bartonella spp. According to the different length of electrophoresis bank, the sample was identified as a species of genus Bartonella other than the positive control. Sequence analysis showed that the nuleotide sequence from the PCR product of primer TIle.455p-TAla.885n was identical to the Bartonella isolated from Yunnan in China. CONCLUSIONS: PCR-based assay provided a simple and rapid means to detect pathogenic Bartonella species in humans and mammalian hosts as well as in arthropod vecters. This study suggested that this pathogenic Bartonella species existed in patients in northern and southern parts of China.

Animals↗

Recommendations for microbial source tracking: lessons from a methods comparison study.

The methods comparison study described in accompanying manuscripts demonstrated the potential value of microbial source tracking (MST) techniques, but also identified a need for method refinement. This paper provides three classes of recommendations to improve MST technology: optimization, development and evaluation. Optimization recommendations focus on library-dependent methods and include improved selection of restriction enzymes or antibiotics, better definition of appropriate library size, selection of target species and choice of statistical pattern-matching algorithms. Methods development recommendations focus on identifying new genomic targets and quantification procedures for library-independent methods. Longer-term methods development recommendations include integration of microarrays and other direct pathogen detection technology with MST. Studies defining host specificity and population dynamics should aid selection of target species during methods development. Evaluation recommendations include enhancements that should be incorporated into future methods comparison studies, along with studies to assess the value of MST results for risk characterization.

Animals↗

Intrauterine infections and birth defects.

Intrauterine infection is an important cause of some birth defects worldwide. The most common pathogens include rubella virus, cytomegaloviurs, ureaplasma urealyticum, toxoplasma, etc. General information about these pathogens in epidemiology, consequence of birth defects, and the possible mechanisms in the progress of birth defects, and the interventions to prevent or treat these pathogens' infections are described. The infections caused by rubella virus, cytomegaloviurs, ureaplasma urealyticum, toxoplasma, etc. are common, yet they are proved to be fatal during the pregnant period, especially during the first trimester. These infections may cause sterility, abortion, stillbirth, low birth weight, and affect multiple organs that may induce loss of hearing and vision, even fetal deformity and the long-term effects. These pathogens' infections may influence the microenvironment of placenta, including levels of enzymes and cytokines, and affect chondriosome that may induce the progress of birth defect. Early diagnosis of infections during pregnancy should be strengthened. There are still many things to be settled, such as the molecular mechanisms of birth defects, the effective vaccines to certain pathogens. Birth defect researches in terms of etiology and the development of applicable and sensitive pathogen detection technology and methods are imperative.

Animals↗

[Desirable role of the clinical laboratory in hospital infection control].

Prevention of hospital-acquired infection is the most important strategy to control infection in terms of the well being of patients and the medical economy. Infection control in hospitals is carried out by the action against hospital-based infection, for which clinical laboratory functions, such as surveillance and outbreak investigation, should be primary responsible. Therefore, it is essential for clinicians to have clinical laboratory rapidly detect pathogenic organisms and provide new and update information on appropriate antibiotics and clinical isolates. It is also desirable for clinical laboratory to collaborate with infection control team (ICT) and link nurse, and provide them with useful information on on-going infection.

Anti-Bacterial Agents↗