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Results for “pathogen detection”

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PubMed1991

High incidence of serum antibodies to Escherichia coli O157 lipopolysaccharide in children with hemolytic-uremic syndrome.

Because the classic hemolytic-uremic syndrome has been etiologically linked to intestinal infections by Escherichia coli O157 and other verotoxin-producing E. coli (VTEC), we examined 22 consecutive children with acute hemolytic-uremic syndrome for the presence of VTEC, using microbiologic methods, and for a specific immune response to O157 lipopolysaccharide in acute-phase and follow-up sera, using the indirect hemagglutination assay and the immunoblot procedure. Of 22 children with enteropathic hemolytic-uremic syndrome, 15 (68%) had evidence of VTEC infection by culture of the pathogen or detection of free verotoxin in the feces, or both. Significantly elevated titers of short-lived agglutinins and IgM class antibodies against the O157 lipopolysaccharide were found in 20 (91%) of 22 patients, but not in two of three patients with non-O157 E. coli isolates or in healthy children or children with diarrhea caused by other enteric pathogens (p less than 0.01). The combined microbiologic and serologic procedures provided evidence for VTEC infection in all 22 patients. The high incidence of anti-O157 lipopolysaccharide antibodies in these patients indicates the predominance and the pathogenic potential of this serogroup. Both serologic techniques proved to be valuable tools to further characterize this form of hemolytic-uremic syndrome. Future studies on the induction of protective immunity seem warranted.

Antibodies, Anti-Idiotypic↗

PubMed2006

[Detection of Yersinia enterocolitica in diarrhea stool by real-time PCR].

OBJECTIVE: To establish a quick and accurate molecular biological method to detect the Yersinia enterocolitica in diarrhea stool as an alterative to the tedious and time-consuming culture methods. METHODS: Real-time PCR, using the primer-probe specific for the yst gene, was applied to detect the pathogenic (7 serotypes) and nonpathogenic (5 serotypes) Yersinia enterocolitica, other species of Yersinia (8 types), and other enteric bacteria (8 types). Additionally, 200 samples of diarrhea stool were examined by culture method too so as to test the consistency of these two methods. RESULTS: Real-time PCR was 100 percent specific for the virulent Yersinia enterocolitica with the detection limits of 10(2) CFU/ml and 10(3) CFU/g for the pure culture and stool sample respectively. Of the 200 samples for comparison, 18 positive samples were successfully examined by both real-time PCR and culture method with a consistency of 100 percent. CONCLUSION: Highly specific for virulent Yersinia enterocolitica without cross reaction to other bacteria, convenient to operate, and rapid to get the result, real-time PCR can be used as a quick method to detect Yersinia enterocolitica from diarrhea stool.

Diarrhea↗

PubMed2003

Analysis of sepsis in allogeneic bone marrow transplant recipients: a single-center study.

We reviewed the records of 235 consecutive recipients of allogeneic bone marrow transplantation (allo-BMT) at our center between February 1983 and October 2000. Sepsis occurred in 25 patients (10.6%) at a median of 10 days (range, 1-280 days) after BMT. Five of the 25 patients (20%) died of sepsis. Pathogens isolated from blood culture were gram-positive cocci in 19 patients, gram-negative rods in 7, fungi in 2, and others in 1 patient. Two pathogens were detected concomitantly in 4 patients. Univariate analysis revealed that risk factors for sepsis were selective gut decontamination using lomefloxacin hydrochloride and nystatin, an unrelated donor, HLA mismatched BMT, and stomatitis. Multivariate logistic regression analysis revealed that an unrelated donor was the only significant independent risk factor, with a relative risk of 5.432. In 12 of 25 patients with sepsis, the pathogens of sepsis were sensitive to antibiotics used for gut decontamination. Selective gut decontamination significantly increased the incidence of sepsis, especially that with gram-positive cocci, but not the mortality rate of sepsis, compared with total gut decontamination using vancomycin. We also found a significant relationship between pathogens isolated from blood culture and those isolated from surveillance cultures of stool, urine, and gargled water in the period before sepsis occurred. The present study revealed an independent risk factor for sepsis (unrelated donor), the feasibility of selective gut decontamination, and the importance of surveillance culture.

Adolescent↗

PubMed2003

Acute infectious diarrhea in children.

INTRODUCTION: Acute infectious diarrhea is a global health problem especially in infants and children, and is a leading cause of morbidity and mortality. The ethiology of acute infectious diarrhea and also biochemical, epidemiological and clinical characteristics of children dying with infectious diarrhea are investigated in this study. METHODS: 201 children, aged from 6 months to 14 years, with acute infectious diarrhea admitted to the Infectious Diseases Clinic in Tuzla in the period from 21st December 1999 to 21st December 2000 were included in the study. RESULTS: Enteropathogens were identified in stool samples in 103 (51.3%) of 201 examined children. Viruses were identified in 51 (25.4%) cases, bacteria in 44 (21.9%), fungi in 3 (1.5%), and parasites in 2 (1%). Rotavirus, a frequent pathogen, was detected in 48 cases (23.9%), followed by Salmonella species in 20 (10%), EPEC in 10 (4.9%), and Shigella species in 9 (4.5%) cases. In this study the authors noticed that the highest morbidity was recorded in children in the first 2 years of life (70.5%), and among rural community (68.4%). The detection of rotavirus decreased with increasing age of cases and peaked in winter and autumn. Blood in stool was most common in children with shigellosis (22.2%). CONCLUSIONS: High percentage of infants and children dying with acute infectious diarrhea presents a serious socio-economic and medical problem in Tuzla region of Bosnia and Herzegovina. Rotavirus is the single most common pathogen in children with infectious diarrhea.

Acute Disease↗

PubMed2006

Sequence-specific, electronic detection of oligonucleotides in blood, soil, and foodstuffs with the reagentless, reusable E-DNA sensor.

The ability to detect specific oligonucleotides in complex, contaminant-ridden samples, without the use of exogenous reagents and using a reusable, fully electronic platform could revolutionize the detection of pathogens in the clinic and in the field. Here, we characterize a label-free, electronic sensor, termed E-DNA, for its ability to simultaneously meet these challenging demands. We find that because signal generation is coupled to a hybridization-linked conformational change, rather than to only adsorption to the sensor surface, E-DNA is selective enough to detect oligonucleotides in complex, multicomponent samples, such as blood serum and soil. Moreover, E-DNA signaling is monotonically related to target complementarity, allowing the sensor to discriminate between mismatched targets: we readily detect the complementary 17-base target against a 50 000-fold excess of genomic DNA, can distinguish a three-base mismatch from perfect target directly in blood serum, and under ideal conditions, observe statistically significant differences between single-base mismatches. Finally, because the sensing components are linked to the electrode surface, E-DNA is reusable: a 30-s room temperature wash recovers >99% of the sensor signal. This work further supports the utility of E-DNA as a rapid, specific, and convenient method for the detection of DNA and RNA sequences.

Biosensing Techniques↗

PubMed2001

Detection of heat injury in Listeria monocytogenes Scott A.

Methods of detecting live pathogens in foods that may be growth inhibited following heat treatment are essential to food safety. Among the techniques available, reverse transcription polymerase chain reaction (RT-PCR) amplification of messenger RNA from heat-injured Listeria monocytogenes Scott A is preferable to direct PCR in an attempt to avoid false positives from dead cells. The RT-PCR has a detection limit of 3 x 10(6) CFU/g, compared to 3 CFU/g for untreated controls, but may not be suitable for the identification of all viable cells. Physically apparent changes in cellular structures from heat injury in L. monocytogenes are expected to result. Ultrastructural analyses did depict notable heat damage as cytoplasmic clearing after 5 min at 60 degrees C. The heat-injured survivors can be readily distinguished from total viable cells using selective media. As a result, combinations of molecular and visual methods including selective media improve detectability of heat-injured, viable L. monocytogenes Scott A.

Colony Count, Microbial↗

PubMed1999

Absence of vertical transmission of infectious salmon anemia virus (ISAV) from individually infected Atlantic salmon Salmo salar.

Atlantic salmon Salmo salar L. eggs were collected from grilse that were individually identified as ISAV-positive based on the detection of pathogen in ovarian fluid by RT-PCR. The eggs were fertilised, disinfected and reared under quarantine conditions. To address the possibility of vertical transmission, fertilised eggs, alevins and parr were screened for the virus by SHK-1 cell culture and RT-PCR. In addition, ISAV-negative parr were injected with homogenates of potentially infected eyed eggs. ISAV was not detected in eyed eggs, alevins or parr. No mortalities occurred among fish injected with the egg homogenates. These observations suggest the absence of a vertical transmission route for ISAV infection.

Anemia↗

PubMed1992

Diagnostic tests for periodontal diseases.

A high level of interest in the development of diagnostic tests capable of detecting factors associated with progressing ("active") periodontal disease has continued in the past year. Notable advances and refinements have occurred in subtraction radiographic techniques and in the development of computer-linked, controlled-force periodontal probes. Development and evaluation of new diagnostic tests have also proceeded at a rapid pace. Particular emphasis has been placed on rapid tests for putative periodontal pathogens and detection in gingival crevicular fluid of host-derived enzymes. An intense effort must be made to determine the clinical value of diagnostic tests in longitudinal studies that use state-of-the-art assessments of disease progression.

Benzoylarginine-2-Naphthylamide↗

PubMed2006

[Etiology, risk factors, and outcome of urinary tract infection].

OBJECTIVES: To determine the incidence and variety of microorganisms of positive urine culture in patients of intensive care units of Kaunas University of Medicine Hospital and to estimate the risk factors for colonization, development of urinary tract infection, and outcome during the period of 2003-2004. MATERIAL AND METHODS: We retrospectively reviewed patients with a positive urine culture with or without clinical symptoms of urinary tract infection admitted to an intensive care unit. RESULTS: The growth of microorganisms was determined in urine of 82 (3.9%) patients: urinary tract infection in 64 (78%) and colonization in 18 (22%) patients. Among pathogens of urinary tract infection rods predominated significantly (p<0.05). No significant difference was found among colonizing microorganisms (p>0.05). E. coli in urine culture was obtained exclusively in cases of urinary tract infection. All patients with a positive urine culture had catheterized urinary bladder (p<0.05). No influence of age, sex, or duration of catheterization alone on the occurrence of urinary tract infection, colonization, or outcome was found (p>0.05). In cases of the same sex, age, and underlying disease of the patients, the risk of urinary tract infection with every day of catheterization significantly increased by 21.7% (p<0.05). Among patients with lethal outcome there was a significant prevalence of rods in urine culture (p<0.05). In survivors, no significant pathogen was detected in urine culture (p>0.05). In cases of the same sex, age, underlying disease, and duration of catheterization, the development of urinary tract infection significantly increased the risk of lethal outcome by 5.5 times (p<0.05). CONCLUSIONS: Positive urine culture was found in 3.9% of patients, three-quarters of them were due to urinary tract infection. Rods were the predominant pathogens in urinary tract infection. E. coli in urinary tract always resulted in urinary tract infection. Catheterization of urinary bladder resulted in urinary tract infection or colonization. Each day of urinary bladder catheterization increased the risk of development of urinary tract infection by 21.7%. Rods in urine culture were associated with lethal outcome. Development of urinary tract infection increased the risk of lethal outcome 5.5 times.

Age Factors↗

PubMed2000

Evaluation of a PCR probe capture assay for the detection of Toxoplasma gondii. Incorporation of uracil N-glycosylase for contamination control.

Toxoplasma gondii is a cyst-forming parasite of clinical relevance in humans primarily because of the neurologic abnormalities it can cause. In some clinical circumstances, it is desirable to detect the pathogen directly. We modified a commercially available Toxoplasma polymerase chain reaction (PCR) probe capture assay by incorporating uracil N-glycosylase (UNG) to prevent carryover amplicon contamination. In addition, UNG inactivation and DNA denaturation were accomplished chemically to simplify the DNA hybridization to the capture probe. The incorporation of UNG effectively eliminated carryover contamination; the probe capture assay showed a log increase in detection sensitivity compared with standard agarose gel electrophoresis. To assess sensitivity and possible inhibition of amplification, different sample types were spiked with Toxoplasma organisms. After DNA extraction and PCR amplification, a sensitivity of 2 tachyzoites for the assay was determined in buffered saline, cerebrospinal fluid (CSF), serum, and amniotic fluid; 20 tachyzoites for whole blood; and 200 tachyzoites for brain tissue. An additional 20 human serum and CSF samples submitted for Toxoplasma serologic testing were run by the PCR method. Of these, only an IgM-positive CSF sample was PCR positive. The Toxoplasma PCR probe capture assay showed good sensitivity and was not substantially inhibited by several different clinically relevant samples.

Amniotic Fluid↗

PubMed2005

New human coronavirus, HCoV-NL63, associated with severe lower respiratory tract disease in Australia.

A new human coronavirus, HCoV-NL63, was associated recently with bronchiolitis. The current study aimed to examine retrospectively stored specimens for the presence of HCoV-NL63 using nested RT-PCR assays targeting the 1a and 1b genes. The study population was composed of patients with acute respiratory disease warranting presentation to Queensland hospitals. HCoV-NL63 was detected in the nasopharyngeal aspirates (NPA) of 16 of 840 specimens representing 766 patients (2%). HCoV-NL63 positive individuals were diagnosed most commonly with lower respiratory tract (LRT) disease (81%). The clinical diagnosis was commonly supported by an abnormal chest X-ray (56%) together with respiratory distress (50%), wheeze (44%), and rales (25%) on first presentation with HCoV-NL63 infection. All patients positive for HCoV-NL63 required admission to hospital. Among 38% of HCoV-NL63 positive specimens a second pathogen was detected. Sequencing of amplicon from gene 1b revealed more than 99% nucleotide homology with the viral type strains while sequencing amplicon from gene 1a permitted the grouping of viral strains. It was shown that HCoV-NL63 is associated with severe LRT disease in an Australian hospital setting during the cooler months of the year. We propose that HCoV-NL63 is a global and seasonal pathogen of both children and adults associated with severe LRT illness.

Adolescent↗

PubMedDate not supplied

A serological survey of rural dogs and cats on the southwestern Canadian prairie for zoonotic pathogens.

A survey for antibodies against agents of plague, tularemia, and Rocky Mountain spotted fever (RMSF), and against Sin Nombre hantavirus (SNV), Bartonella henselae and B. clarridgeiae was conducted in the summer of 1995 using serum from rural dogs and cats living in the vicinity of four public parks in southeastern Alberta and southwestern Saskatchewan. Antibodies to all pathogens were detected in all survey areas. Overall prevalence rates were 0.075 for Yersinia pestis, 0.089 for Francisella tularensis, 0.025 for Rickettsia rickettsii (dogs only), and 0.029, 0.178 and 0.186 for SNV, B. henselae and B. clarridgeiae, respectively (cats only). This serological survey of rural dogs and cats was more sensitive and efficient than previous surveys based on collection and culture of rodents and ectoparasites. All six pathogens appear endemic to the region. Surveillance for plague, tularemia, RMSF and SNV, and management of associated public risks should be done in endemic regions.

Animals↗

PubMed1995

Polymerase chain reaction detection of small round-structured viruses from two related hospital outbreaks of gastroenteritis using inosine-containing primers.

Two outbreaks of gastroenteritis in the UK which occurred nine days apart at Lymington and Southampton hospitals were investigated. The clinical and epidemiological features of both outbreaks were characteristic of small round-structured virus (SRSV) infection with rapid onset of diarrhoea and/or nausea and vomiting and propagation of the outbreaks by secondary spread. SRSV particles were observed by immune electron microscopy (EM) in 60% of faecal samples from both outbreaks and no other pathogens were detected. The index case for the second outbreak was a patient who was admitted with diarrhoea and vomiting after being discharged from Lymington hospital during the first outbreak. The possibility that the two outbreaks were caused by the same strain of SRSV was investigated by the polymerase chain reaction (PCR). New inosine-containing PCR primers were designed to amplify the RNA polymerase region of SRSV cDNA from genetic groups I and II. The PCR using the group II primers achieved a higher detection rate for SRSVs in faecal samples (68% of samples positive from both outbreaks) than immune EM. SRSVs were not detected using the group I primers or using conventional degenerate PCR primers. The nucleotide sequences of PCR amplicons from both outbreaks were identical providing molecular epidemiological evidence for the involvement of a single SRSV strain. Comparison of the RNA polymerase region of this virus with the equivalent regions of genetic group I (69.4-75.0% amino acid identify) and genetic group II (88.9-100% amino acid and 77.1-88.1% nucleotide identity) SRSVs revealed that the causative SRSV was a distinct member of genetic group II.

Amino Acid Sequence↗

PubMed2003

A highly sensitive immuno-PCR assay for detecting Group A Streptococcus.

A highly sensitive hybrid assay, based on immuno polymerase chain reaction (immuno-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques, was developed for the detection of pathogenic Group A Streptococcus (Strep A). Cells were disrupted by sonication and then coated onto the walls of Maxisorp microtiter plates. Next, biotinylated anti-Group A monoclonal antibody (mAb) was bound to the antigen and then linked, via a streptavidin (STV) bridge, to biotinylated reporter DNA. After extensive washing, the denatured reporter DNA was transferred to PCR tubes, amplified, electrophoresed, and used as the signal for detection of bacteria. The minimum detection limit of this assay is the equivalent of approximately one one-thousandth of a Streptococcus pyogenes cell, even in the presence of 100,000 Escherichia coli cells. The combination of multiple antigens per cell and PCR amplification provides the extreme sensitivity in this immuno-PCR assay. No cross-reaction was found with other Streptococcus species. We also directly linked the anti-Group A monoclonal antibody to DNA using succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The sensitivity using directly linked antibody-reporter DNA was approximately 10 cells. Because this assay could be adapted for detection of many different bacteria in a variety of sample types, we tested the potential for interference from substances that could be present in clinical, food, and environmental samples. Sonicated meat or human plasma did not inhibit detection; however, extracts of concentrated soil samples were somewhat inhibitory. This highly specific, sensitive, and robust assay could be applied to clinical detection of Group A Streptococcus and serves as a model for other immuno-PCR assays.

Antibodies, Monoclonal↗

PubMed2001

A prevalence survey for zoonotic enteric bacteria in a research monkey colony with specific emphasis on the occurrence of enteric Yersinia.

Transmissible pathogenic and opportunistic zoonotic enteric bacteria comprise a recognized occupational health threat to exposed humans from non-human primates (NHPs). In an effort to evaluate the occurrence of selected enteric organisms with zoonotic and biohazard potential in a research colony setting, we performed a prevalence study examining 61 juvenile and young adult rhesus macaques participating in a transplant immunology project. Primary emphasis was directed specifically to detection of pathogenic enteric Yersinia, less well-documented and reported NHP pathogens possessing recognized significant human disease potential. NHPs were surveyed by rectal culture during routine health monitoring on three separate occasions, and samples incubated using appropriate media and specific selective culture methods. Enteric organisms potentially transmissible to humans were subcultured and identified to genus and species. Significant human pathogens of the Salmonella/Shigella, Campylobacter, and enteric Yersinia groups were not isolated throughout the survey, suggesting prevalence of these organisms may generally be quite low.

Animal Technicians↗

PubMed1999

Demonstration of the pathogenic effect of point mutated keratin 9 in vivo.

A wild type keratin 9 (K9) cDNA and a point mutated keratin 9 cDNA were injected subcutaneously into mouse skin. The hemagglutinin tag staining of the wild type K9 cDNA injected specimens mainly showed a homogeneous pattern, whereas the point mutated K9 cDNA injected specimens mainly showed a granular pattern in the suprabasal cells. Double staining of K9 and the endogenous keratin revealed the incorporation of de novo synthesized K9 into the keratin network. These results demonstrate that (1) a naked DNA transfection into mouse skin can detect the pathogenic changes of point mutated keratin in vivo and (2) the keratin 9 mutation disrupts the keratin network formation in the suprabasal cells in vivo.

Animals↗

PubMed2006

Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever.

Rickettsia typhi and Rickettsia felis are the etiologic agents of murine typhus and flea-borne spotted fever, respectively. We have constructed two quantitative real-time polymerase chain reaction (qPCR) assays to detect these pathogenic rickettsiae. The qPCR assays were developed utilizing unique sequences of the R. typhi and R. felis outer membrane protein B genes (ompB) to design the specific primers and molecular beacon probes. The assays were found to be species-specific and did not yield false-positive reactions with nucleic acid from other rickettsiae, orientiae, neorickettsiae or unrelated bacteria. In addition, the assays were sensitive enough to detect three target sequence copies per reaction and were capable of detecting R. typhi and R. felis nucleic acid in the cat flea, Ctenocephalides felis. These results demonstrate that two sensitive and specific qPCR assays have been successfully developed to detect and enumerate R. typhi and R. felis.

Animals↗

PubMed2000

Solid phase microextraction/gas chromatography of Salmonella-infected beef.

Eight strains of Salmonellae were incubated in TSB culture medium at 37 degrees C for 24 h. Volatile compounds derived from the bacteria were collected using solid-phase microextraction fibers and then applied to gas chromatography (GC). Similarity analysis of the GC patterns thus obtained could separate these strains on principal component similarity (PCS) scattergrams. Five major food-related pathogenic bacteria and 10 other bacteria (including one Salmonella strain) were also classified by growing in the same medium. It is then proposed to utilize this approach to improve the GC/PCS method of Nakai et al. [Nakai, S.; Wang, Z. H.; Dou, J.; Nakamura, S.; Ogawa, M.; Nakai, E.; Vangerstoep, J. J. Agric. Food Chem. 1999, 47, 576-583] that has been developed for screening safe foods by detecting bacteria contaminated foods. Inoculating food samples pre-enriched through preliminary incubation into a culture medium and then subjecting to the GC/PCS method after secondary incubation enhances the detectability of pathogenic bacteria.

Animals↗