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PubMed2003

Molecular detection and typing of fungal pathogens.

A major goal of molecular testing is to develop a cost-effective as well as sensitive and specific assay that can detect microbial DNA in clinical samples early in the course of disease. Additionally, the ability to analyze the genetic relatedness of fungi on a timelier basis using molecular methods will have a positive impact on epidemiologic investigating. As technology advances, it seems apparent that commercially available molecular assays will become available in the near future for the management of patients with suspected fungal infections.

DNA, Fungal↗

PubMed2002

Chlamydia pneumoniae in carotid artery atherosclerosis: a comparison of its presence in atherosclerotic plaque, healthy vessels, and circulating leukocytes from the same individuals.

BACKGROUND AND PURPOSE: There is growing clinical and experimental evidence that infections with Chlamydia pneumoniae might contribute to the development and progression of atherosclerosis. However, studies detecting the pathogen in atherosclerotic lesions examined either only atherosclerotic vessels or control vessels without atherosclerosis obtained from a different group of individuals. We analyzed atherosclerotic plaques of the carotid artery, samples of apparently healthy greater saphenous veins, and circulating leukocytes from the same individual patients for the presence of C pneumoniae. METHODS: From each of 46 patients undergoing carotid endarterectomy for symptomatic carotid artery stenosis, these samples were analyzed by nested polymerase chain reaction for C pneumoniae-specific DNA. Furthermore, we determined IgA and IgG titers specific for the pathogen and plasma levels of C-reactive protein in these patients. RESULTS: C pneumoniae DNA was detected in 86.9% of the leukocytes and in 82.6% of the atherosclerotic plaques but in only 6.5% of the saphenous veins. In 85% of patients who also had leukocytes positive for C pneumoniae, the atherosclerotic plaques were positive and the saphenous veins were negative. The presence of C pneumoniae-specific DNA in leukocytes significantly coincided with the presence of the respective DNA in the plaques of the carotid arteries (P=0.0002). No association between the presence of C pneumoniae and specific IgA or IgG levels was seen. C-reactive protein levels were significantly higher in patients with chlamydia-positive atherosclerotic plaques and with positive leukocytes than in patients with negative plaques of the carotid arteries or negative leukocytes, respectively (P<0.01, P<0.05). CONCLUSIONS: Our observation of >80% incidence of C pneumoniae in atherosclerotic plaques of the carotid artery does not prove causality between an infection with the pathogen and the development of atherosclerosis. It must be emphasized, however, that >90% of apparently healthy saphenous veins were negative for C pneumoniae. Given the structural and functional differences between veins and arteries, careful interpretation of our results regarding a possible causative role of C pneumoniae seems warranted.

Aged↗

PubMed1999

Isolation and detection of Listeria spp, Salmonella spp and Yersinia spp using a simultaneous enrichment step followed by a surface adhesion immunofluorescent technique.

The use of a rapid surface adhesion immunofluorescent (SAIF) technique for the isolation of three pathogens using a single enrichment step from broth and enriched meat cultures was investigated. Buffered peptone water (BPW, 225 ml) was inoculated with Listeria monocytogenes, Salmonella enteritidis and Yersinia enterocolitica to a level of 10 cfu ml(-1) and incubated overnight at 30 degrees C. Minced beef samples (25 g) were inoculated with the three pathogens to a level of 100 cfu g(-1) and incubated overnight at 30 degrees C in BPW (225 ml). Pathogens were isolated by surface adhesion to a polycarbonate membrane which was immersed in the enriched culture. The pathogens were detected using membrane counts (rinse and immunofluorescent) and standard plate counts. It was noted that the attachment of the three pathogens to the membrane was considerably enhanced when compared with single inoculum experiments and the reasons and implications of this for the rapid method are discussed. A small selection of naturally contaminated retail samples (n = 60) was tested for the presence of L. monocytogenes and S. enteritidis using a simultaneous enrichment step with SAIF detection and a standard cultural method of detection. A good correlation was found between both methods for L. monocytogenes (r2 = 0.91, RSD = 0.26) and S. enteritidis (r2 = 0.89, RSD = 0.28). This indicates that simultaneous analysis of these two pathogens could be carried out using the SAIF assay.

Animals↗

PubMed1999

Detection of low numbers of pathogenic Yersinia enterocolitica in environmental water and sewage samples by nested polymerase chain reaction.

Isolation of pathogenic Yersinia enterocolitica from water and sewage by traditional culture techniques is time-consuming and subsequent differentiation between pathogenic and non-pathogenic strains can be difficult and unreliable. A nested polymerase chain reaction (PCR) procedure was used for the detection of low numbers of Y. enterocolitica in spiked samples from natural surface sources with variable background flora ranging from oligotrophic water to sewage. Water and sewage samples were filtered and filters enriched overnight in a non-selective medium. Nested PCR conducted on enriched broth, prepared by use of a rapid and simple preparation step consisting of centrifugation, proteinase K treatment and boiling, enabled the detection of 8-17 cfu 100 ml-1 water with background levels of up to 8700 heterotrophic organisms ml-1 and 10,000 cfu coliform organisms 100 ml-1 water. The analysis can be completed within 2-3 d and should be a significant tool in monitoring environmental waters and drinking water sources for the presence of pathogenic Y. enterocolitica.

Environmental Monitoring↗

PubMed1999

Identification of bacterial pathogens in patients with endophthalmitis by 16S ribosomal DNA typing.

PURPOSE: To determine whether sequence analysis of 16S ribosomal DNA (rDNA) can be used to detect bacterial pathogens in patients with postoperative endophthalmitis. METHODS: In 10 eyes of 10 patients, vitreous specimens were collected for culture and rDNA typing. Variable segments of each ribosomal DNA specimen were amplified by polymerase chain reaction (PCR), sequenced, and aligned by BLAST, a computer alignment program, against sequences in GenBank at the National Institutes of Health. RESULTS: Specimens were available from five eyes with bacterial endophthalmitis diagnosed by Gram stain or culture. Amplified 16s rDNA sequences from the eyes of three patients were identical to microbiologic results. Polymerase chain reaction results were negative in two cases in which unusual organisms were detected. All five control specimens from patients with nonbacterial endophthalmitis or uveitis were PCR negative. Approximately 48 to 72 hours are required under ideal conditions for final species identification with this ribosomal typing technique. CONCLUSIONS: 16S rDNA typing shows potential as a relatively rapid technique for identifying bacteria in vitreous samples.

Bacteria↗

PubMed2001

Comparison of a multiplex reverse transcription-PCR-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens.

A multiplex reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA; Hexaplex; Prodesse Inc., Waukesha, Wis.) was used for the simultaneous detection of human parainfluenza virus types 1, 2, and 3, influenza virus types A and B, and respiratory syncytial virus types A and B. One hundred forty-three respiratory specimens from 126 patients were analyzed by RT-PCR-EHA, and the results were compared to those obtained by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of 143 (5.6%) samples were positive by viral culture and/or IF. Eight samples were positive by both RT-PCR-EHA and conventional methods, while nine samples were RT-PCR-EHA positive and viral culture and IF negative. Eight of the nine samples with discordant results were then independently tested by a different multiplex RT-PCR assay for influenza virus types A and B, and all eight proved to be positive. In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was able to detect more positive samples, which would otherwise have been missed by routine methods, we suggest that this multiplex RT-PCR-EHA provides a highly sensitive and specific means of diagnostic detection of major respiratory viruses.

Adolescent↗

PubMed1995

Detection of bacteraemia by the continuously monitoring BacT/Alert system.

AIMS: To analyse a continuously monitoring blood culture system with respect to the time to detection of various groups of organisms, their clinical importance, and the relative efficacy of the aerobic and anaerobic bottles. METHODS: Four thousand blood cultures were monitored and the information relating to the positive cultures was noted and analysed. RESULTS: Four hundred and seventy seven blood cultures were detected as positive, 81% (387/477) of which were detected within 48 hours. The most pathogenic organisms were detected in the shortest period, less pathogenic later and those generally regarded as contaminants last. Clinically important isolates were also detected earlier. Many positive blood cultures were detected in only one bottle of the set, even those regarded as clinically important. CONCLUSIONS: The management of continuously monitoring blood culture systems could be improved by considering time to detection trends. Clinicians should be aware of the relatively rapid detection of clinically important, positive blood cultures in relation to patient treatment.

Automation↗

PubMed2005

Evaluation of detection methods for screening meat and poultry products for the presence of foodborne pathogens.

Rapid and molecular technologies such as enzyme-linked immunosorbent assay (ELISA), PCR, and lateral flow immunoprecipitation can reduce the time and labor involved in screening food products for the presence of pathogens. These technologies were compared with conventional culture methodology for the detection of Salmonella, Campylobacter, Listeria, and Escherichia coli O157:H7 inoculated in raw and processed meat and poultry products. Recommended protocols were modified so that the same enrichment broths used in the culture methods were also used in the ELISA, PCR, and lateral flow immunoprecipitation assays. The percent agreement between the rapid technologies and culture methods ranged from 80 to 100% depending on the pathogen detected and the method used. ELISA, PCR, and lateral flow immunoprecipitation all performed well, with no statistical difference, compared with the culture method for the detection of E. coli O157:H7. ELISA performed better for the detection of Salmonella, with sensitivity and specificity rates of 100%. PCR performed better for the detection of Campylobacter jejuni, with 100% agreement to the culture method. PCR was highly sensitive for the detection of all the foodborne pathogens tested except Listeria monocytogenes. Although the lateral flow immunoprecipitation tests were statistically different from the culture methods for Salmonella and Listeria because of false-positive results, the tests did not produce any false negatives, indicating that this method would be suitable for screening meat and poultry products for these pathogens.

Animals↗

PubMed2005

Antimicrobial susceptibilities of potential bacterial pathogens in adults with acute respiratory tract infections Prospective Epidemiological Network Investigating Community-acquired Infection SurveiLLance In Nagasaki (PENICILLIN) Study.

The prevalence and the antimicrobial susceptibilities of bacterial pathogens in acute upper respiratory tract infections (AURTIs) is not clear. We conducted a prospective community-based multicenter study in 45 centers across Nagasaki, Japan, between December 2001 and April 2002. We examined the nasopharyngeal or throat isolates in 930 adult outpatients with AURTIs. Potential bacterial pathogens, including Streptococcus pyogenes (from 6.8% patients), S. pneumoniae (5.3%), S. milleri group (2.0%), Staphylococcus aureus (26.0%), Moraxella catarrhalis (7.4%), Haemophilus influenzae (17.6%), and Klebsiella pneumoniae (1.2%), were isolated from 51.8% of the patients. Penicillin nonsusceptible phenotype accounted for 44.9% of S. pneumoniae isolates. Among Staphylococcus aureus isolates, 9.1% were methicillin-resistant. In H. influenzae isolates, 6.7% produced beta-lactamase and 23.2% were beta-lactamase-negative ampicillin-resistant. The results indicated that the potential bacterial pathogens were detected in the remarkable population of the adults with AURTIs and the current antimicrobial susceptibilities of such pathogens.

Acute Disease↗

PubMedDate not supplied

Haptoglobin and serum amyloid A in milk from dairy cows with chronic sub-clinical mastitis.

New tools are needed to detect chronic sub-clinical mastitis, especially in automatic milking systems. Haptoglobin and serum amyloid A (SAA) are the two most sensitive bovine acute phase proteins, and their concentrations increase in milk from cows with clinical mastitis and in milk from cows with experimentally induced chronic sub-clinical Staphylococcus aureus mastitis. The aim of this study was to further evaluate the potential for haptoglobin and SAA in milk as indicators of chronic sub-clinical mastitis. Quarter milk samples were collected from 41 cows with a mean composite milk somatic cell count (CSCC) above 300,000 cells/mL during at least two months prior to sampling. Quarter milk samples were also taken from eleven cows with a mean CSCC below 80,000 cells/mL during at least two previous months. These samples were analysed for haptoglobin, SAA, adenosine triphosphate (ATP) activity and bacterial growth. The samples were grouped according to their ATP, haptoglobin and SAA status. ATP+ samples had ATP > 2 x 10(-10) mol/mL, Hp+ and SAA+ samples had detectable levels of haptoglobin (> or = 0.3 mg/L) and SAA (> or = 0.9 mg/L), respectively. In udder quarter samples from healthy cows, 42 out of 44 samples belonged to the ATP-Hp-SAA- group. Among cows with chronic sub-clinical mastitis, the ATP+Hp+SAA+ group contained 66 out of 164 samples while 44 samples belonged to the ATP+Hp-SAA- group. Detectable levels of haptoglobin and SAA were found in 92 and 80 samples, respectively. Growth of udder pathogens was detected in 28 samples and Staphylococcus aureus was the most common bacteria. In conclusion, haptoglobin and SAA concentrations below the detection limit were considered as good indicators of healthy udder quarters. A substantial variation in haptoglobin and SAA concentrations in milk was observed in udder quarters with chronic sub-clinical mastitis.

Adenosine Triphosphate↗

PubMed1989

Specific gene probe for detection of biotyped and serotyped Listeria strains.

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.

Bacterial Typing Techniques↗

PubMed1995

The applications of the polymerase chain reaction in the life sciences.

The polymerase chain reaction (PCR) allows the in vitro amplification of DNA fragments starting with tiny amounts of biological sample and oligonucleotide primers derived from sequence data. Since the technique is fast and easy, PCR has taken the DNA-technology to the routine laboratoria. We present a survey of the following applications of PCR: 1) The amplification of gene fragments as fast alternative of cloning. 2) The modification of DNA fragments. 3) The sensitive detection of pathogenic microorganisms, if desired followed by an accurate genotyping. 4) DNA analysis of arachaeological specimens. 5) The detection of mutations relevant for inherited diseases, malignant transformation or tissue typing. 6) The analysis of genetic markers for forensic applications, for paternity testing and for the mapping of hereditary traits. 7) The species-specific amplification of DNA segments between interspersed-repeat elements. 8) The study of gene expression.

Animals↗

PubMed1993

Detection and identification of a pathogenic marine Mycobacterium from the European seabass Dicentrarchus labrax using polymerase chain reaction and direct sequencing of 16S rDNA sequences.

Mycobacteriosis has become a major concern for the commercial mariculture of the European sea bass Dicentrachus labrax in Israel. The disease remains asymptomatic for a long time, is virtually impossible to eradicate with antibiotics, stunts the growth of the fish and renders the fish unmarketable. The pathogen was identified as Mycobacterium marinum by direct sequencing and analysis of approximately 600 bp of the pathogen ribosomal encoding DNA (rDNA). The polymerase chain reaction technique was evaluated as a diagnostic tool for detecting the infection in D. labrax and found to be highly specific and sensitive.

Animals↗

PubMed2005

Methods to characterize lymphoid apoptosis in a murine model of autoreactivity.

The immune system is shaped by the random generation of lymphocytes followed by apoptosis of self-reactive cells, a process termed negative selection. The survival of these pathogenic cells in the periphery can elicit autoreactivity. We describe the development of a biomarker assay for the detection of pathogenic subpopulations of lymphoid cells in adult non-obese diabetic (NOD) mice based on disease-specific alterations in spontaneous or triggered cell death. Utilizing improved methods of cell separations, two distinct lymphoid cell subpopulations with increased susceptibility to apoptosis were identified and quantified. A subpopulation of CD8+ T cells that constitutes approximately 3-7% of the total CD8+ T cell population underwent apoptosis on exposure to low concentrations of TNF-alpha. Such cells were exclusively detected only in NOD mice with histologic signs of active autoreactivity. The non-T cell compartment of NOD immune system, although resistant to TNF-alpha-induced apoptosis, contained a subpopulation of B cells with spontaneous death by culture alone. The refined detection of small numbers of lymphoid cell subsets with quantifiable differences in apoptosis provides a possible immune biomarker for monitoring disease activity or treatment interventions.

Animals↗

PubMed2000

[Evaluation of the usefulness of selected virulence markers for the identification of virulent Yersinia enterocolitica strains. I. Phenotypic markders associated with Plasmid pYV].

The species Yersinia enterocolitica includes either pathogenic or non-pathogenic strains. Therefore it is necessary to differentiate virulent bacilli from other. It is well known that pathogenic strains of Y. enterocolitica bearing virulence associated plasmid called pYV, which could be demonstrated by its isolation or detected by the presence of specific, phenotypic properties directly related with this plasmid. The aim of the presented paper was to check the ability of some phenotypic virulence markers associated with pYV, to detection of pathogenic Y. enterocolitica strains. In the presented work 152 (130 carrying pYV) clinical strains of Y. enterocolitica O3 isolated mainly from stool were examined for the presence of phenotypic virulence markers such as: calcium dependency, Congo-red binding, autoagglutination and agglutination with Mangifera indica extract. Both first features were detected parallel, on the same plate, using CRMOX (Congo-red, Magnesium Oxalate) agar. The detection of the tested markers in the examined strains was compared with the presence of virulence plasmid. The obtained results confirmed the observations done by other authors that Y. enterocolitica strains, in which bacilli bearing the virulence plasmid predominate, exhibit all tested phenotypic properties whereas the plasmid-cured isogenic strains show no one of these features. Therefore all the tested markers could be useful for detection of virulent Y. enterocolitica strains directly isolated from patients. The most useful virulence markers in bacteriological study seems to be calcium dependency and Congo-red binding, examined together by the use of CRMOX agar, because they confirm the presence of the virulence plasmid by parallel detection of two physiologically different features associated with this plasmid. In addition CRMOX agar allows for the examination rough strains while agglutination tests do not.

Bacterial Proteins↗

PubMedDate not supplied

Studies of bacterial, rotaviral and Cryptosporidium etiology of acute diarrheal diseases in hospitalized children.

657 hospitalized children with acute diarrheal disease were studied for bacterial and rotaviral etiology. Cryptosporidium presence was followed in 123 children. Intestinal pathogens were detected in 195 (29.6%) cases: 132 (20.3%) enterobacteria, 47 (7.1%) rotaviruses, 4 (3.2%) Cryptosporidium sp. and 12 (1.8%) combined infections. Among enterobacteria, E. coli was the most frequent (10% cases) with enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic (EHEC) and enteroinvasive (EIEC) groups. Other isolated pathogens were Salmonella--21 (3.1%), C. jejuni/coli--13 (1.9%), Shigella--3 (0.9%), Y. enterocolitica O3--1 (0.1%). Among opportunistic pathogenic enterobacteria, the following were isolated: Kl. pneumoniae--24 (3.7%), Enterobacter species--4 (0.6%), Providencia alcalifaciens--1 (0.1%). Aeromonas hydrophila was isolated in 1 child. Combined infections were detected in 12 children: 10--enterobacteria + rotaviruses associations and 2--pathogenic enterobacteria associations.

Acute Disease↗

PubMed2004

Evaluation of a multiplex reverse transcriptase PCR ELISA for the detection of nine respiratory tract pathogens.

BACKGROUND: A multiplex reverse transcription (RT) polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) was previously developed to detect nine different microorganisms: enterovirus (EV), influenza virus type A (IVA) and type B (IVB), respiratory syncytial virus (RSV), parainfluenzavirus type 1 (PIV1) and type 3 (PIV3), adenovirus (AV), Mycoplasma pneumoniae (Mpn), Chlamydia pneumoniae (Cpn) in a single test. These organisms do not usually colonize the respiratory tract of humans, but, if present, it may be assumed they are involved in respiratory disease. OBJECTIVES AND STUDY DESIGN: The m-RT-PCR-ELISA was tested on (i) culture supernatants of unknown contents, (ii) by determining the analytical sensitivity of 10-fold serial dilutions of culture supernatants and (iii) by determining clinical sensitivity in a retrospective study on 411 clinical specimens. The specimens were re-tested in parallel by m-RT-PCR-ELISA versus the gold standard culture and immunfluorescence, and versus individual RT-PCR. RESULTS: (i) The 9-valent m-RT-PCR-ELISA shows 83% to 100% concordant results on 103 culture supernatants containing different organisms. (ii) The analytical sensitivity was as follows: higher sensitivity of the 9-valent m-RT-PCR-ELISA in comparison to culture in the cases of PIV3, IVA and IVB (factor 10) and AV and EV (factor 100), and lower sensitivity in case of RSV and PIV1 (factor 10). (iii) The agreement with the gold standard in the kappa statistic was excellent for RSV (kappa = 0.937), IVA (kappa = 0.940), very good for PIV1 (kappa = 0.914), IVB (kappa = 0.907) and satisfactory for PIV3 (kappa = 0.410). For AV, EV and Mpn the m-RT-PCR-ELISA preliminary could be qualified as very good, based on the data derived on culture supernatants. Information about the validity for Cpn is limited. CONCLUSION: The m-RT-PCR-ELISA is a feasible, sensitive and specific method for detection of a broad spectrum of organisms. It is suitable for individual as well as epidemiological diagnosis.

Adenoviridae↗

PubMed1998

Comparison of clinically directed, disease specific, and syndromic protocols for the management of genital ulcer disease in Lesotho.

OBJECTIVE: To evaluate two protocols for the syndromic management of genital ulcer disease (GUD) in Lesotho, southern Africa and to compare the performance of these protocols with that of a conventional disease specific approach. METHODS: A cross sectional study was conducted among consecutive patients with GUD attending an STD clinic in Maseru, Lesotho. The clinical diagnoses were made by using predefined criteria at the initial visit before the performance of laboratory tests. Attempts were made to detect the specific aetiology of the genital ulcers using PCR assays and syphilis serology. The results of PCR assays and syphilis serology were used as the gold standard against which the performance of the management approaches were applied. RESULTS: Of 100 patients initially recruited into the study, Haemophilus ducreyi infection was detected in 56%, herpes simplex virus in 26%, Treponema pallidum in 23%, and lymphogranuloma venereum in 7%. No pathogens were detected in 6% of patients. 17% of patients had mixed infections. Sensitivity, specificity, positive and negative predictive values of the three management protocols for GUD were compared after applying each to the study population. Theoretically, the lowest correct treatment rate would have been obtained by using the disease specific protocol (62%) compared with more than 90% in both syndromic management protocols. Considerable overtreatment for primary syphilis would occur following application of both syndromic protocols. This would be the result of the overdiagnosis of chancroid, in particular the misdiagnosis of genital herpes as chancroid, which would receive treatment for syphilis unnecessarily. The HIV seroprevalence among these patients was 36%. A significantly higher rate of HIV seropositivity was detected among the patients with herpes simplex virus infection when compared with those patients having other causes of genital ulcer disease (58% v 27%; odds ratio 3.73; 95% CI 1.26-11.26; p = 0.01). CONCLUSIONS: Poor sensitivity, specificity, and predictive values were recorded when the disease specific protocol was applied to the study population. In contrast, the syndromic management protocols provided adequate treatment for more than 90% of patients with GUD. Protocol C, which identified a minority of cases of genital herpes, was found to have an advantage when compared with protocol B (all patients with genital ulcer disease treated for both syphilis and chancroid) in that 29% of genital herpes cases would receive appropriate counselling.

Chancroid↗