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PubMedDate not supplied

Specificity of the BAX polymerase chain reaction system for detection of the foodborne pathogen Listeria monocytogenes.

The polymerase chain reaction (PCR) can be used for rapid and specific detection of foodborne pathogens. One commercial kit, the Qualicon BAX system uses PCR to detect Listeria monocytogenes in enrichment cultures derived from food and environmental samples. The specificity and sensitivity of the BAX system for detecting L. monocytogenes were characterized by using both pure and mixed cell cultures, and optimal conditions for production of cell lysates were determined. The BAX system was highly specific for L. monocytogenes, and no interference was seen in the presence of either other Listeria species or microbes from other genera. The assay detected L. monocytogenes at 10(5)-10(6) colony-forming units/mL. This sensitivity is adequate for detecting viable cells after enrichment but prevents false-positive signals from nonviable cells.

Electrophoresis, Polyacrylamide Gel↗

PubMed1992

Polymerase chain reaction-gene probe detection system specific for pathogenic strains of Yersinia enterocolitica.

The polymerase chain reaction technique was used to develop a rapid diagnostic assay for detection of pathogenic Yersinia enterocolitica strains. The assay targeted a stretch of 163 bp of the yst gene and could be applied to both pure cultures and crude DNA extracted from feces. The defined primer pair amplified the targeted sequence from only pathogenic strains and fecal samples seeded with the serotype O:3 strain of Y. enterocolitica, whereas neither nonpathogenic strains nor normal stools yielded any amplified fragments. Of the other Yersinia species and non-Yersinia species tested, only two strains of Y. kristensenii yielded the same amplified product. A 20-mer oligonucleotide probe specifically hybridized within the amplified yst fragment of Y. enterocolitica but did not hybridize with the amplified yst fragment of Y. kristensenii by Southern and dot blot hybridizations. This confirms the reliability of this diagnostic assay in both clinical and epidemiological studies. The availability of the extracted DNA for the polymerase chain reaction was checked by simultaneous amplification of a part of the 16S rDNA and the yst gene. The entire diagnostic assay, including a simplified technique for DNA extraction, the amplification process, and gel electrophoresis, could be completed within 1 working day, which is better than the time required for the time-consuming traditional techniques used in clinical laboratories.

Base Sequence↗

PubMed2006

[DNA microarrays and their application in detecting and identifying intestinal pathogens].

DNA microarrays offer many advantages of high throughout, automation, rapid detection, and so on. Therefore, this technology had been used in many fields such as molecular epidemiology of bacteria, microbial gene identification, disease mechanism, gene mutation, gene expression identification, DNA sequencing and medicine screening etc. The assays for identifying pathogens using DNA microarrays reported aboard recently are introduced. The application of DNA microarrays in detecting and identifying intestinal pathogens mainly includes three aspects: the identification of toxin and characteristic genes of pathogens, the identification of bacterial DNA or RNA directly, the simultaneous detection of a large number of intestinal pathogens with the target - gene of ribosomal RNA. Because of its high efficiency, DNA microarrays is superior to other biological method. Obviously DNA microarrays technology may be useful in identifying intestinal pathogens and have a wide prospect.

Animals↗

PubMed2003

Rapid detection and identification of tomato vascular wilt pathogens using a DNA array.

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or V. dahliae, are devastating diseases of tomato (Lycopersicon esculentum Mill.) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We demonstrate that by using this array these pathogens can be detected within 24 h from complex substrates like soil, plant material, and samples as they are collected by tomato growers in their greenhouses.

DNA, Fungal↗

PubMed2003

Detection and identification of pathogens and host DNA in unfed host-seeking Ixodes ricinus L. (Acari: Ixodidae).

In this study, we have developed molecular methods for the identification of reservoir hosts of sylvatic tick-borne zoonoses. The methods are based on the analysis of the blood meal remnant in the tick gut and include detection of pathogens and identification of the host origin of the blood meal. For host identification, a universal primer pair was used to amplify part of the vertebrate 18S rRNA gene followed by reverse line blot hybridization using subgroup-specific probes. Analyses of DNA from whole blood of vertebrates identified the correct subgroup of a broad range of vertebrate species (e.g., Ruminantia, Leporidea, Canidae, Murinae, Arvicolinae, Insectivora, Galliformes, Passeriformes) using probes based on the 18S rDNA sequences. Host DNA in the remnants of larval blood meals was detected in the gut of Ixodes ricinus nymphs maintained under natural conditions up to 9 mo after molting. For pathogen identification, a multiplex polymerase chain reaction was used that targeted parts of the 18S rRNA gene of piroplasm protozoa, the 16S rRNA gene of bacteria, and the intergenic spacer of the Borrelia burgdorferi genospecies complex. The utility of both methods was demonstrated under laboratory conditions by detecting Babesia microti (Franca) and gerbil DNA in 3-mo-old I. ricinus nymphs that had fed on B. microti-infected gerbils as larvae, and under field conditions by analyzing unfed ticks that were collected in a forest. The field study showed that the majority of ticks had fed on ruminants or birds and few on rodents, which is in accord with our knowledge of the fauna in this forest. Few pathogens were detected but the discovery of Borrelia valaisiana and B. burgdorferi s.s. in ticks that had fed on deer and Borrelia afzelii in a tick that had fed on a bird raises questions about the mode of transmission of these spirochetes and possibly about their host specificity.

Animals↗

PubMed2004

Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescence.

The simultaneous detection of Staphylococcus aureus, Listeria monocytogenes, and Salmonella spp. has been approached by a new multiplex PCR-based procedure followed by capillary gel electrophoresis with laser-induced fluorescence detection (multiplex-PCR-CGE-LIF). As compared to slab gel electrophoresis, the use of CGE-LIF improved from 10- to 1000-fold the sensitivity of the multiplex PCR analysis, allowing the detection of 2.6 x 10(3) cfu mL(-1) of S. aureus, 570 cfu mL(-1) of L. monocytogenes, and 790 cfu mL(-1) of Salmonella in artificially inoculated food, without enrichment. Following 6 h of enrichment, as low as 260, 79, and 57 cfu mL(-1) of S. aureus, L. monocytogenes, and Salmonella, respectively, were detected. The CGE-LIF method is shown to be reproducible, providing relative standard deviation (RSD) values lower than 0.8% for analysis time and lower than 5.8% for peak areas. The multiplex-PCR-CGE-LIF proved a powerful analytical tool to detect various food pathogens simultaneously in a fast, reproducible, and sensitive way.

Electrophoresis, Capillary↗

PubMed1998

Bacteria and viruses that cause respiratory tract infections during the pilgrimage (Haj) season in Makkah, Saudi Arabia.

OBJECTIVE: To determine the incidence and type of RTI-causing bacteria and viruses during a period of epidemic infections. METHOD: A total of 395 sputum specimens and 761 throat swabs were collected during the 1991 and 1992 pilgrimage seasons (Haj to Makkah Al-Mukarama, Saudi Arabia) from patients referred to one hospital and three dispensaries with symptoms of respiratory tract infections. All 761 throat swabs of both Haj seasons were also screened for the presence of viral pathogens with monoclonal antibodies specific for 7 viruses known to cause respiratory infections. RESULTS: Bacterial pathogens were detected in 118 (29.9%) specimens. During the 1991 Haj season Haemophilus influenzae was the most frequent bacterial pathogen detected (10%), followed by Klebsiella pneumoniae (5.2%), Streptococcus pneumoniae (4.8%), Staphylococcus aureus (3.8%) and Streptococcus pyogenes (2.4%). In the 1992 Haj season Klebsiella pneumoniae was predominant (15.1%), followed by Haemophilus influenzae and Streptococcus pneumoniae (12.3%). Screening of all sputum specimens for acid-fast bacteria showed that the overall incidence rate of tuberculosis was 1%. Cultures from the 761 throat swabs were largely negative for bacteria except for Streptococcus pyogenes isolated from 7 patients. Viruses were detected in 148 (19.5%) specimens with influenza A and adenovirus being the most common viruses. CONCLUSION: The pattern of virus prevalence in the 1991 and 1992 pilgrimage seasons was identical: influenza A and adenovirus predominated. Thus these two viruses should be targeted in future prophylactic measures.

Antibodies, Monoclonal↗

PubMed2006

Detection and identification of intestinal pathogens in clinical specimens using DNA microarrays.

The detection and identification of intestinal pathogens is critical for clinical patient diagnosis and antimicrobial therapy. No currently available assays with DNA microarrays can simultaneously detect and identify multiple intestinal pathogens, because there is no appropriate method for choosing target probes. To solve the problem we have experimented for facilitating screening of specific probes and developed a rapid (<3h) and reliable assay for simultaneous detection of intestinal pathogens using two universal PCR primers to amplify two variable regions of bacterial 16S and 23S ribosomal DNA (rDNA) genes, and then applied to DNA microarrays, hybridization between probes and amplicons occurred. Through this idea for screening of probes the assay was successful in discriminating 15 genera or species of intestinal pathogens. The limit of detection was approximately 10(3)CFU/mL for one species of pathogen and 10(5)CFU/mL for six species pathogens existing simultaneously in stool. When this assay was applied directly to identify 99 clinical specimens, 80(80.8%) were correctly analyzed, including four with mixed pathogens; 8(8.08%) received negative results due to no corresponding probes in this array and 11(11.11%) belonging to our targets were misidentified due to low-level pathogens and other factors. This approach is also convenient to obtain specific and proper probes while establishing assays for the applications in other aspects using DNA microarrays. In addition, the more species may be added to this system easily and endlessly by screening of candidate target probes in order to increase the power of simultaneous detection.

Bacterial Infections↗

PubMed1994

Evaluation of two commercial blood culture media for the detection of blood-borne pathogens.

To determine their ability to detect blood-borne pathogens, the blood culture media BCB Release (Becton Dickinson, USA) and Signal (Oxoid, UK) were evaluated on a total of 5,122 blood culture sets. Each of the two bottles was inoculated with an equal amount of blood from 2,262 patients at bedside (2.3 cultures per patient). In the laboratory, agar-coated paddles were attached to the BCB Release bottles and the Signal device was mounted onto the Signal bottles. Both systems were incubated at 35 degrees C for seven days. A total of 608 (11.9%) sets were positive, from which 549 pathogenic microorganisms could be isolated. These could be grown significantly (p < 0.0001) more often from the BCB Release broth (n = 483) than from the Signal system (n = 384). Mainly staphylococci, Escherichia coli and yeasts were isolated more often from the BCB Release, anaerobes were more often detected from the Oxoid Signal. The time to positivity was significantly shorter (p < 0.0001) with the BCB Release. The Signal system proved a valuable complement to the BCB Release broth.

Adolescent↗

PubMedDate not supplied

[Cryptosporidium sp. in children with diarrhea in Cuba].

During the period from May to September 1987 stools from 200 children (aged 2 months to 4 years) with diarrhoea at Children's Hospital in Havana City were tested for enteropathogens. Three stool samples collected from each patient on alternative days were examined for Cryptosporidium oocysts by direct wet mounts, concentration by the method of Ritchie (formol-ether sedimentation) and by modified Ziehl-Neelsen staining technique. Total prevalence of intestinal parasitoses was as high as 24.5%. Cryptosporidium infection was found in 8% (16 children). Cryptosporidium was the second most commonly detected enteric pathogen overall after Lamblia intestinalis (identified in 10%). In the overwhelming majority of patients Cryptosporidium was the only detectable pathogen (13 cases of monoinfection). In the youngest children Cryptosporidium was the commonest parasite. It is noteworthy that all children who excreted the Cryptosporidium oocysts were exclusively bottle-fed.

Animals↗

PubMed1993

Diagnostic workup strategies for patients with HIV-related chronic diarrhea. What is the end result?

The intensity of diagnostic workup of patients with human immunodeficiency virus (HIV)-related chronic diarrhea is controversial. In the ideal setting in which an enteric pathogen is detected with minimal evaluation (stool tests) and in which specific treatment clears the diarrhea, eradicates the pathogen, and improves the patient's quality of life, the need for diagnostic workup is clear. However, problems frequently occur in the evaluation and treatment of patients that preclude such a straightforward approach. They are (a) failure to detect enteric pathogens; (b) detection of organisms of uncertain significance; (c) lack of effective treatment; (d) the presence of a severe coexisting illness that is the major determinant of the patient's outcome; and (e) lack of evidence that detecting enteric pathogens leads to improvement in broad patient outcomes, such as quality of life. I discuss these problems and examine the two opposing diagnostic workup strategies--minimal and intensive evaluation--that have been advocated. My main conclusion is that both approaches fall short, and that clinicians lack the information needed to guide clinical decision making. I urge investigators to analyze the full effects of alternative diagnostic interventions on broad patient outcomes so that clinical guidelines can be developed to assist the evaluation of patients.

AIDS-Related Opportunistic Infections↗

PubMed2004

New chromogenic plating media for detection and enumeration of pathogenic Listeria spp.--an overview.

In recent years a number of selective chromogenic plating media for pathogenic Listeria spp. have been developed and marketed. Their advantages are direct detection and enumeration of pathogenic Listeria spp. utilizing cleavage of substrates by the virulence factor phosphatidylinositol-phospholipase C (PI-PLC) and, to a lesser extent, by phosphatidylcholin-phospholipase C (PC-PLC). There are two groups of such media: the first utilizes cleavage by PI-PLC of L-alpha-phosphatidyl-inositol, forming a white precipitation zone around the colony, combined with the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside for detection of beta-d-glucosidase, which occurs in all Listeria spp. All Listeria spp. produce turquoise colonies on these media which include ALOA , CHROMagar Listeria, BBL CHROMagar Listeria, and OCLA. The second group of media utilizes 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate, forming blue-turquoise colonies of pathogenic Listeria spp. and white colonies of non-pathogenic Listeria spp. BCM trade mark Listeria monocytogenes plating medium, Rapid'L.mono and LIMONO-Ident-Agar belong to this group. Selective chromogenic L. monocytogenes plating media offer the attraction of rapid economic detection and enumeration of pathogenic Listeria spp. within 24 or 48 h of incubation at 36+/-1 degrees C. This overview summarises the characteristics of these chromogenic plating media, reviews important evaluations, and focuses on replacement of conventional by these chromogenic plating media, particularly for applications in the food industry.

Chromogenic Compounds↗

PubMed1991

Development and application of a nonradioactive nucleic acid hybridization system for simultaneous detection of four potato pathogens.

cDNA clones of potato virus X (PVXcp strain), potato virus Y (PVYo strain), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTV) were used separately or combined for the detection of the corresponding RNAs in extracts of infected plants. A general method for the rapid preparation of RNA extracts without use of organic solvents (i.e. phenol) was developed for this purpose. Plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and protoplast samples, as well as vector insect extracts, were dot-blotted onto nylon or nitrocellulose membranes, subjected to sandwich nucleic acid hybridization with non-labelled specific single-stranded DNA probes followed by a biotin-labelled second step hybridization probe. Each probe was virus-specific but not strain-specific. Healthy or non-related plant extracts developed very faint or no signals. Sensitivity was tested by slot-blot hybridization. Detection levels were between 1.5 to 6 pg of viral nucleic acids and between 20 to 50 times more sensitive than standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The assay developed was tested with material that was prepared for processing in the field (combination of fresh sap with extraction solution) and tested under simple laboratory conditions for detection. It was also successfully employed for screening of germplasm for virus resistance, detection of pathogens in vector insects, plantlets grown in vitro and in more sophisticated quantitative determinations of viral replication in artificially inoculated plants and protoplasts.

Cloning, Molecular↗

PubMed2006

Enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests.

The advantages of nucleic acid amplification tests (NAT) over conventional methods for the detection of pathogens in lower respiratory tract samples have not been established. NAT for respiratory pathogens were performed on 439 endotracheal tube (ETT) and bronchoalveolar lavage (BAL) samples. A potential pathogen was detected in 87 samples. Of 22 samples that tested positive by conventional methods, 15 tested positive for the same pathogen by NAT, 1 tested positive for a different pathogen, 2 had co-infections identified only by NAT, and 4 tested negative by NAT. An additional 73 pathogens were detected by NAT in 65 samples including 30 pathogens that were missed by conventional methods (19 adenovirus, 6 respiratory syncytial virus, 3 parainfluenza virus 1-4, 2 influenza A), 41 pathogens not routinely identified by conventional methods in most laboratories (23 rhinovirus, 8 human coronavirus OC43, 5 human metapneumovirus (hMPV), 2 human coronavirus 229E, 2 human coronavirus NL63, 1 Chlamydophila pneumoniae) and 2 pathogens from samples where no respiratory virus testing was requested (1 influenza A, 1 parainfluenza virus). Four of 52 patients who had multiple BAL samples submitted on the same day had negative and positive results by NAT on different samples. NAT improves detection of potential pathogens from ETT and BAL samples.

Adolescent↗

PubMed1979

A novel technique for detecting IgA coated potentially pathogenic microorganisms in the human intestine.

This paper describes a novel method for detecting immunoglobulin A (IgA) coated potentially pathogenic microorganisms (PPMs) in the human intestine. Essentially, the technique consists of 2 phases: one in which IgA coated bacteria are detected by immunofluorescence and a second in which these bacteria are subcultured in situ and subsequently identified. In this way transient bacteria are differentiated from resident bacteria. These results show that the resident bacteria are coated with IgA. Resident microorganisms are always highly concentrated in the digestive tract. These results strengthen the hypothesis that only the high antigen concentrations achieved by a large number of resident bacteria are capable of IgA induction.

Antibodies, Bacterial↗

PubMed1990

Microbiological agents associated with childhood diarrhoea in the dry zone of Sri Lanka.

Between March and July, 1987, faecal specimens from 371 diarrhoeal children reporting to four hospitals in the district of Kurunegala, Sri Lanka and from 121 controls with other diseases were investigated for enteric pathogens. All specimens were tested for Shigella spp, Salmonella spp and enteropathogenic Escherichia coli (EPEC). In addition, parasitic pathogens, heat labile enterotoxigenic Escherichia coli (ETEC LT), Campylobacter and rotavirus were sought among subsamples of the cases and controls. One or more pathogens were detected in the faecal specimens of 53.7% of the diarrhoeal children and 19.6% of the controls (P less than 0.001). Rotavirus was the most frequently identified pathogen. Cases (21.9%) were more likely to be positive for rotavirus than controls (6.5%) (p less than 0.05). Among children with diarrhoea the peak prevalence of rotavirus occurred in the 6-11 months age group. Shigella spp were the second most frequently isolated pathogens and the single most common cause of diarrhoea in children over two years of age. Again, cases (16.4%) were more likely to be infected than controls (1.7%) (p less than 0.001). Salmonella spp were isolated from 7.5% of diarrhoeal children and 3.3% of the controls (p greater than 0.10). Other pathogens had prevalence rates of less than 5%. Vomiting was associated with rotavirus diarrhoea (p less than 0.001), and fever with shigellosis (p = 0.02). In view of the different transmission routes and characteristics of the most important pathogens detected in this study it appears unlikely that a single environmental intervention can solve the public health problem posed by diarrhoeal diseases in Sri Lanka.

Acute Disease↗

PubMed2006

Multiple detection of food-borne pathogenic bacteria using a novel 16S rDNA-based oligonucleotide signature chip.

There have been many attempts to develop sensitive and accurate techniques for the detection and diagnosis of pathogenic bacteria using nucleic acid-based technology. To achieve efficient multiple detection of seven selected food-borne pathogens, we assessed the respective 16S rDNA pathogen specific sequences using an oligonucleotide-based signature array. Strategic optimal design of specific capture probes was achieved by using the characteristic first variable region. To assess the specificity of this pathogen detection system, we employed a two-step experimental strategy. Under conditions established through experiments with chemically synthesized model targets comprising both conserved and variable regions of 16S rDNA, we confirmed the validity of this system using real 16S rDNA targets. Detection with real targets was successfully performed using our system, and better specificity was obtained compared to experiments with model targets. Moreover, the subtypes of Vibrio pathogens were successfully classified. We developed a two-dimensional visualization plot tool for positive control and specific spots, which allowed facile and minute differentiation between spot intensities. Repeated array formats were employed to ensure experimental uniformity, and included the statistical p-value criterion for pathogen discrimination. The present results thus indicate that our novel oligonucleotide-based signature chip detection system can be employed for the effective detection of multiple pathogens.

Bacteria↗

PubMedDate not supplied

Steatorrhea: a common manifestation in patients with HIV/AIDS.

Multiple infectious causes of diarrhea are known in patients with HIV/AIDS. Maldigestion and malabsorption have been reported in patients with HIV/AIDS and may be independent of infectious etiologies. Among ambulatory patients with HIV/AIDS, we examined the prevalence of fat malabsorption (steatorrhea). Sixty-one patients with unexplained diarrhea (defined as > 2 stools/d) and/or weight loss despite adequate caloric intake (and without clinical evidence of chronic pancreatitis) were evaluated in our outpatient Gastroenterology-Nutrition Clinic between March 1, 1993, and July 1994. Patients were instructed by a dietitian to follow a > or = 100 g/d fat diet for 24 h before submitting a stool sample for qualitative (or quantitative) fecal fat determination. Forty-five patients, 32 with ongoing diarrhea and 13 without diarrhea, submitted stool samples. Twenty-two of 45 patients (49%) had qualitative or quantitative steatorrhea, 16/32 with diarrhea (50%) and 6/13 patients without diarrhea (46%). Thirty of 32 patients with diarrhea had had extensive microbiologic and/or endoscopic evaluations. Only 9 patients had a detectable intestinal pathogen, 5 patients had cytomegalovirus (4 treated), 4 patients had cryptosporidia (3 treated), and 1 patient had microsporidia. Steatorrhea, as determined by abnormal qualitative fecal fat, is detectable in nearly 50% of patients with HIV/AIDS. Fat malabsorption appears to be a primary defect in these patients independent of detectable pathogens. Assessment of fat malabsorption should be considered in patients with unexplained weight loss or diarrhea before extensive evaluation for opportunistic infections.

Acquired Immunodeficiency Syndrome↗