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PubMed1995

Polymerase chain reaction for detection of Chlamydia pneumoniae in gargled-water specimens of children.

The objective of the present study was to establish the occurrence of Chlamydia pneumoniae by direct detection in gargled-water specimens obtained from 193 children suffering from acute or chronic respiratory infections. Specimens were analyzed by an indirect immunofluorescence test (IIF), a genus-specific antigen enzyme immunosorbent assay (EIA) and the polymerase chain reaction (PCR). The pathogen was detected in three children by PCR only. As underlying disease, chronic obstructive bronchitis resistant to therapy was reported. In two of the children, the presence of pneumonia could be verified by X-ray. With a detection threshold of target DNA obtained from two inclusion forming units (IFU), the PCR proved clearly more sensitive than EIA becoming positive at levels of 100 IFU and above. No interpretable results could be obtained for the IIF.

Adolescent↗

PubMed1987

Occurrence of multiple antibiotic resistance and R-plasmids in gram-negative bacteria isolated from faecally contaminated fresh-water streams in Hong Kong.

The bacterial populations of six freshwater streams in populated areas of the Hong Kong New Territories were studied. There is considerable faecal contamination of these streams, with coliform counts as high as 10(5) c.f.u./ml and the contaminating organisms show a high prevalence of antibiotic resistance and multiple resistance. With direct plating of water samples onto antibiotic-containing media, an average of 49% of the gram-negative bacteria were ampicillin-resistant, 3% chloramphenicol-resistant and 1% gentamicin-resistant. At individual sites resistance to these drugs was as high as 98%, 8% and 3% respectively. More than 70% of strains were resistant to two or more antibiotics, 29% to five or more and 2% to eight or more. A total of 98 patterns of antibiotic resistance were detected with no one pattern predominating. Twenty-eight gram-negative bacterial species were identified as stream contaminants. Escherichia coli was the commonest bacterial species isolated and other frequent isolates were Enterobacter sp., Klebsiella sp. and Citrobacter sp., but no enteric pathogens were detected. The greatest prevalence of resistance and multiple resistance was associated with the heaviest contamination by E. coli. Analysis of selected stream isolates revealed multiple plasmid bands arranged in many different patterns, but multiple antibiotic resistances were shown to be commonly mediated by single transferable plasmids. Faecally-contaminated freshwater streams in Hong Kong may be reservoirs of antibiotic resistance plasmids for clinically-important bacteria.

Animals↗

PubMed1997

Urinary recovery of lactulose compared to D-xylose absorption kinetics in HIV patients with diarrhea and weight loss.

Using a kinetic model of D-xylose absorption, we have previously shown that there is severely impaired absorption of D-xylose in HIV patients with diarrhea and weight loss. The absorptive defect is characterized by an increased rate constant for nonabsorptive loss of D-xylose, Ko, and a decreased absorptive rate constant, Ka, and is unrelated to histology or the presence of pathogens. It is not known if there is also abnormal paracellular transport in these patients. We have extended our observations in these patients by including a measurement of paracellular transport, lactulose absorption. Nine HIV patients with chronic diarrhea, weight loss, and no detectable intestinal pathogens, two healthy volunteers, and three non-HIV patients with chronic diarrhea (two functional and one with scleroderma) were enrolled. Of the nine HIV patients, six had diminished bioavailability of D-xylose, F (range: 19-52%, normal >70%), and elevated rate constant for nonabsorptive loss, Ko (range: 0.54-1.35/hr, normal <0.353/min). Four of the six also had decreased Ka (range: 0.09-0.36/hr, normal >0.634/min). Only one of these six had increased lactulose recovery (3.51%, normal <0.5%). Two of three patients with normal kinetic parameters of D-xylose absorption had increased lactulose urinary recovery (1.92%, 2.61%). In conclusion, lactulose absorption is increased in some patients with HIV-related diarrhea who have normal D-xylose absorption, suggesting a paracellular mechanism for diarrhea in some patients with AIDS enteropathy.

Diarrhea↗

PubMed2006

[Toll-dependent and toll-independent innate antiviral immunity].

Until recently, adaptive immunity and cytotoxic T cells were considered as the only essential components of the antiviral defence arsenal. Additional data that do not rule out the crucial role of these cells in the clearance of viral pathogens have, however, recently emerged. They indicate that innate immune cells such as macrophages, dendritic cells, gammadelta T cells as well as natural killer (NK) cells play a primordial role in this mechanism. It is now well established that innate immune cells can detect various pathogens (bacteria, viruses, fungi or parasites) very rapidly and respond to their presence through the activation of specific receptors. Once activated, these molecules trigger several signalling cascades that culminate in the establishment of very potent defence mechanisms. In addition, cytokines produced during this initial response are essential for the activation of the adaptive immune response which will add specificity and memory to the system. Among the innate immune receptors, attention has focused on the Toll-like receptors (TLR) and many reports indicate that some of the TLRs are clearly involved in defence against viral pathogens. However, new molecules, acting independently from any TLR, have recently been discovered. They define a second antiviral pathway which is presently the subject of intense research. In this article, we will review the role of the different molecules involved in each pathway within the framework of innate antiviral defence.

DEAD Box Protein 58↗

PubMed1998

16S ribosomal DNA typing for identification of pathogens in patients with bacterial keratitis.

The identification of pathogens in patients with bacterial keratitis remains problematic because standard diagnostic tests are negative for 40 to 60% of patients. A cross-sectional study was undertaken to determine if PCR and sequence analysis of 16S ribosomal DNA (rDNA) could be used to detect bacterial pathogens in patients with keratitis. Corneal specimens were collected for culture and rDNA typing. Variable segments of each rDNA specimen were amplified by PCR, sequenced, and aligned with the sequences in GenBank. Eleven patients had microbiologically documented bacterial keratitis, while 17 patients had keratitis due to other causes. Nine (82%) of 11 bacterial keratitis patients were PCR positive; each sequencing result matched the culture results. Seventeen (100%) patients with nonbacterial keratitis were PCR negative. Our data suggest that 16S rDNA typing holds promise as a rapid alternative to culture for identifying pathogens in patients with bacterial keratitis.

Adult↗

PubMed2003

Predominance of Ehrlichia ewingii in Missouri dogs.

To investigate the species distribution of Ehrlichia present in Missouri dogs, we tested 78 dogs suspected of having acute ehrlichiosis and 10 healthy dogs. Blood from each dog was screened with a broad-range 16S rRNA gene PCR assay that detects known pathogenic species of Ehrlichia and ANAPLASMA: The species was determined by using species-specific PCR assays and nucleotide sequencing. Ehrlichia antibody testing was performed by using an indirect immunofluorescence assay with Ehrlichia chaffeensis as the antigenic substrate. The broad-range assay detected Ehrlichia or Anaplasma DNA in 20 (26%) of the symptomatic dogs and 2 (20%) of the asymptomatic dogs. E. ewingii accounted for 20 (91%), and E. chaffeensis accounted for 1 (5%) of the positives. Anaplasma phagocytophilum DNA was detected in one dog, and the sequences of regions of the 16S rRNA gene and the groESL operon amplified from the blood of this dog matched the published sequences of this organism. Antibodies reactive with E. chaffeensis were detected in 14 (67%) of the 21 PCR-positive dogs and in 12 (19%) of the 64 PCR-negative dogs. Combining the results of PCR and serology indicated that 33 (39%) of 85 evaluable dogs had evidence of past or current Ehrlichia infection. We conclude that E. ewingii is the predominant etiologic agent of canine ehrlichiosis in the areas of Missouri included in this survey. E. canis, a widely recognized agent of canine ehrlichiosis, was not detected in any animal. The finding of E. ewingii in asymptomatic dogs suggests that dogs could be a reservoir for this Ehrlichia species.

Animals↗

PubMed1991

Use of electron microscopy in examination of faeces and rectal and jejunal biopsy specimens.

The stools and rectal biopsy specimens of 44 patients with AIDS and diarrhoea were examined by culture, light microscopy, and electron microscopy. In 13 patients examination of rectal biopsy material and faecal samples showed no pathogen, but in two of these, microsporidiosis was found by electron microscopical examination of jejunal biopsy specimens. This organism was also identified electron microscopically in one of the further five jejunal biopsy samples taken from patients with a known cause of diarrhoea. Blastocystis hominis infection was identified electron microscopically in six patients, all of whom had cryptosporidiosis additionally seen by light microscopy. Four of these six patients remained well for long periods, with only moderate diarrhoea, and follow up showed no evidence of blastocystis infection. In only four of 11 patients found to have cryptosporidium in their stools at light microscopy were organisms found at electron microscopy. Viral inclusions were only identified at electron microscopy in one of 10 patients with an opportunistic viral infection seen at light microscopy (cytomegalovirus n = 7, herpes simplex virus n = 3). No additional viral pathogens were detected in either stools or rectal biopsy material by electron microscopy. It is concluded that routine electron microscopic examination of stool samples or rectal biopsy material taken from patients with AIDS and diarrhoea is unnecessary and does not increase the yield of potential pathogens compared with standard microbiological techniques and histology.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Recovery of Listeria monocytogenes from vacuum-sealed packages of frankfurters: comparison of the U.S. Department of Agriculture (USDA) food safety and inspection service product composite enrichment method, the USDA Agricultural Research Service (ARS) product composite rinse method, and the USDA-ARS package rinse method.

This study compared three methods for the recovery of Listeria monocytogenes from commercially prepared and vacuum-packaged frankfurters that were inoculated with a five-strain mixture of this pathogen at averages of 22 and 20,133 CFU per package over three trials. The presence and levels of the pathogen were determined by (i) the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) product composite enrichment method, involving the selective enrichment of a 25-g composite of product and the subsequent plating of this product onto selective agar plates; (ii) the USDA Agricultural Research Service (ARS) product composite rinse method, involving the rinsing of a 25-g composite of product with 0.1% peptone water and the subsequent plating of a portion of the rinse fluid directly onto selective agar plates; and (iii) the USDA-ARS package rinse method, involving the use of 25 ml of 0.1% peptone water to rinse the entire contents of a package and the subsequent plating of a portion of the rinse fluid directly onto selective agar plates. For packages inoculated with 20,133 CFU. L. monocytogenes was recovered at a frequency (percentage of packages positive) of 100% by each of the three methods. The pathogen was recovered at efficiencies (percentages of recovery of L. monocytogenes) of 43 and 94% with the USDA-ARS product rinse method and the USDA-ARS package rinse method, respectively. For packages inoculated with 22 CFU, L. monocytogenes was recovered at frequencies of 17, 10, and 100% by the USDA-FSIS product composite enrichment method, the USDA-ARS product composite rinse method, and the USDA-ARS package rinse method, respectively. The pathogen was recovered at efficiencies of 20 and 95% with the USDA-ARS product composite rinse method and the USDA-ARS package rinse method, respectively. In a related study, the USDA-ARS package rinse method was the only method that detected the pathogen in 60 packages from each of five brands of frankfurters purchased from local grocery stores. These data establish that the USDA-ARS package rinse method is markedly more sensitive, as well as demonstrably more rapid and facile, than either the approved USDA-FSIS product composite enrichment method or the USDA-ARS product composite rinse method in determining the presence or absence of L. monocytogenes and establishing the levels of the pathogen that may be on the surface of ready-to-eat foods such as frankfurters.

Animals↗

PubMedDate not supplied

Detection of PCR products from Mycobacterium avium subspecies Paratuberculosis using oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

A pool of five oligonucleotides has been used to detect the pathogenic organism Mycobacterium avium subspecies paratuberculosis in PCR-amplified DNA from ruminants. The oligonucleotides were labelled at the 5'-end with three dinitrophenyl reporter groups and hybridised to the target DNA, which was fixed to a nylon membrane by ultraviolet irradiation. Colourimetric detection of the PCR product was carried out using an anti-DNP antibody conjugated to horseradish peroxidase or to alkaline phosphatase. Detection with alkaline phosphatase was more sensitive than with horseradish peroxidase but, in both cases, the PCR product could be easily detected. The DNP labelling system offers an economic and effective alternative to biotin, digoxigenin or fluorescein for the detection of PCR-amplified DNA.

Bacterial Typing Techniques↗

PubMed1993

Culture negative cytotoxin positive stools in community acquired diarrhoea.

In a significant proportion of cases of community acquired diarrhoea no recognised pathogen is detected. Verocytotoxin producing Escherichia coli (VTEC) are associated with haemorrhagic colitis and the haemolytic uraemic syndrome but their role in community acquired diarrhoea is not fully understood. Using a method of toxin enhancement in mixed faecal culture, 175 stools negative on culture for recognised pathogens were tested for the presence of cytotoxin and 28% were found to be positive. Nine were neutralised by anti-VT1, 29 by anti-VT2, and eight by neither. No control stool samples yielded any cytotoxin. VTEC should be considered as a causative agent in sporadic community acquired diarrhoea.

Adolescent↗

PubMed1995

Environmental virology: from detection of virus in sewage and water by isolation to identification by molecular biology--a trip of over 50 years.

Environmental virology began with efforts to detect poliovirus in sewage and water more than 50 years ago. Since that time, cell-culture methods useful for detection of enteroviruses have been replaced by molecular biology techniques for detection of pathogens (hepatitis A and E viruses, caliciviruses, rotaviruses, and astroviruses) that do not grow in cell culture or grow with great difficulty. Amplification of viral nucleic acid using the polymerase chain reaction (PCR) is the current preferred method. PCR or RT-PCR (to detect RNA viral genomes) is rapid, sensitive, specific, and quantitative. Method shortcomings include potential inhibition by substances in some environmental samples and an inability of test results to distinguish between infectious and noninfectious virus. Current questions involving use of PCR/RT-PCR tests for public health purposes include: What is the public health significance of a positive test, and should direct tests for viruses replace current public health-monitoring programs?

DNA, Viral↗

PubMed2007

From molecular diagnostics to personalized testing.

Gene-based molecular diagnostics is changing the practice of medicine and will continue to do so for the foreseeable future. The major underlying principle of these diagnostic tests is the use of specific nucleic acid sequences as surrogates; amplification of the surrogate markers enables the detection of pathogens or disease-related gene mutations. Gene targets can be amplified by target-, probe- or signal-based methods. Combined use of nucleic acid amplification and enzyme-linked immunosorbent assay with methods such as immuno-polymerase-chain reaction allows us to detect protein at femtogram (10(15) g) levels. A variety of choices are available for the detection of amplified amplicons with the fluorophore-linked nanoparticles as the most sensitive markers. The unique advantages of using covalently-linked nanoparticles include the detection of single molecules, the ability to enrich molecules of interest with unprecedented detection sensitivity (up to zeptogram levels, 10(21) g) and the flexibility of multiple functionalization. Automation appears to be the current trend for high-volume molecular testing of infectious diseases. Molecular profiling of various diseases using genomic or proteomic approaches opens up a molecule wonderland with promise and emergence of new molecular testing that will likely impact the practice of medicine to a greater degree in the future. The future of molecular-based testing and the journey toward personalized testing will be discussed.

Animals↗

PubMed1999

Norwalk-like virus and bacterial pathogens associated with cases of gastroenteritis onboard a US Navy ship.

Acute gastroenteritis is a potential cause of substantial morbidity in U.S. military personnel during deployment. This study investigated the microbial causes of diarrhea in U.S. troops on exercises in Southeast Asia aboard the U.S.S. Germantown from March through May 1996. A total of 49 (7%) patients with diarrhea reported to sick call during a 3-month deployment involving 721 personnel. Diarrheal samples from 49 patients were subjected to bacterial and parasitologic examination, but sufficient samples from only 47 of 49 were available for analysis of the presence of Norwalk-like virus (NLV). Of the 49 diarrhea cases, 10 (20.4%) appeared to be due to bacterial etiology alone, 10 (20.4%) due to bacteria and the prototype Taunton agent (TNA), 11 (22.4%) due to TNA only, and 4 (8.0%) due to parasites. Norwalk-like virus RNA was present in 21 (45%) of 47 stool samples from the diarrhea cases, 10 with bacterial etiologies and 11 without bacterial or parasitic etiologies. No pathogen was detected in 14 (29%) of the cases. Four of the controls showed the presence of parasitic organisms. Of the 11 cases in which enterotoxigenic Escherichia coli was isolated, 8 were positive for colonization factor antigen (CFA/IV), and 3 were CFA-negative. The bacterial pathogens tested were all susceptible to gentamicin, and furadantin, but were resistant to ceftriaxone and norfloxacin, including 75% of the Campylobacter spp. These data support the view that the major cause of diarrhea for troops deployed in this geographic area is most likely NLVs.

Acute Disease↗

PubMed1998

Antigen-specific immune response of porcine T lymphocytes to various pathogens.

The importance of swine in agriculture has resulted in a substantial increase in research efforts on the swine immune system during the past few years. A better knowledge of the porcine immune system is required before improved vaccination strategies, the design of more efficient vaccines and breeding for disease resistance will be able to contribute to a reduction in the extensive economic losses caused by the disabling effects of viral, bacterial and parasitic infections. T lymphocytes play a central role in the antigen-specific immune response to the various pathogens. To detect and characterise porcine T lymphocytes, monoclonal antibodies (mAbs) were raised against different leucocyte differentiation antigens and classified for specificity in two international workshops. These mAbs have enabled detailed studies to be made on specific cell populations involved in the porcine immune response to pathogens, on T lymphocytes and on the peculiarities of porcine T lymphocyte sub-populations: extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-TCR gamma delta+ T cells. Furthermore, these reagents and the increased knowledge of the immune system have allowed studies of the interactions of T lymphocyte sub-populations with regard to different pathogens and the role which these play in infections.

Animals↗

PubMed2002

Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR.

A rapid multiplex PCR (m-PCR) method that allows the simultaneous detection, in a single tube, of six commonly encountered waterborne pathogens is developed. The target genes used were: the aerolysin (aero) gene of Aeromonas hydrophila, the invasion plasmid antigen H (ipaH) gene of Shigella flexneri, the attachment invasion locus (ail) gene of Yersinia enterocolitca, the invasion plasmid antigen B (ipaB) gene of Salmonella typhimurium, the enterotoxin extracellular secretion protein (epsM) gene of Vibrio cholerae and a species-specific region of the 16S-23S rDNA (Vpara) gene of Vibrio parahaemolyticus were used as the gene targets. Multiplex PCR using the six pairs of primers produced specific amplicons of the expected sizes from mixed populations of reference bacterial strains in seawater and from pure cultures. The m-PCR assay was specific and rapid, with a turnaround time of < 12 h. The detection limit of the assay for the bacterial targets was estimated at 10(0)-10(2) cfu. Multiplex PCR analysis was performed on 19 seawater samples collected around Hong Kong and the results indicated significant levels of four bacterial pathogens at several sites where primary sewage wastes are discharged, and the levels of which showed no correlation with E. coli counts. Overall, both laboratory and field validation results demonstrated that the m-PCR assay developed in this study could provide a cost-effective and informative supplement to conventional microbiological methods for routine monitoring and risk assessment of water quality.

Bacteria↗

PubMed1996

Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR.

To assess the risks from viral contamination of drinking-water supplies, there is a clear need for methods to directly detect viral pathogens. In this study, we developed a broad-spectrum immunocapture method for concentration and purification of enteric viruses. The method involved indirect antibody capture (AbCap) of intact viruses followed by release of virion genomic RNA and reverse transcriptase PCR for amplification and oligoprobe hybridization for detection. The procedure involved concentrating enteric viruses from large volumes of water by standard filtration-elution techniques with IMDS filters and 1 liter of 1% beef extract-0.05 M glycine (BE/G) as an eluate. The BE/G eluate was concentrated and purified by polyethylene glycol (PEG) precipitation, Pro-Cipitate (a commercially available protein precipitating reagent) precipitation, and a second PEG precipitation to a volume of approximately 500 mu l. Aliquots of the second PEG precipitate were further processed by RNA extraction, AbCap, or cell culture analysis for infectious viruses. The AbCap method was applied to 11 field samples of fecally contaminated surface water. Of the 11 samples, 9 were positive for enteric viruses by AbCap method 4 of 11 samples were positive for enteric viruses by direct RNA extraction of a small aliquot of the second PEG concentrate; and 4 of 11 samples were positive for enteric viruses by measurement of cell culture infectivity. The results of enteric viruses were compared with those for standard bacterial and coliphage indicators of fecal contamination.

Animals↗

PubMed2005

Ichthyosis bullosa of Siemens: its correct diagnosis facilitated by molecular genetic testing.

Ichthyosis bullosa of Siemens (IBS, MIM 146800) is a unique congenital ichthyosis characterized by mild epidermal hyperkeratosis over flexural areas, blister formation and the development of superficially denuded areas of hyperkeratotic skin. It is clinically difficult to distinguish severe IBS from mild bullous congenital ichthyosiform erythroderma (BCIE, MIM 113800). In the current literature, 19 IBS families with keratin 2e (K2e) mutations have been reported, despite only five IBS families having been reported before the first identification of K2e mutation in 1994. We studied four patients from three Japanese IBS families. They had previously been misdiagnosed as having BCIE before the correct diagnosis was made after mutation detection. To detect the pathogenic mutations, we performed direct sequencing of the entire coding regions of KRT2E encoding K2e in the patients and healthy family members. K2e mutations, a 1469T-->C transition (L490P) and a 1477G-->A transition (E493K) within the conserved 2B helix termination motif of the rod domain were detected in the families and the definite diagnosis of IBS was made in the four cases. The present results indicate that IBS is not such a rare entity as was previously thought, and accurate diagnosis is now available by mutation analysis.

Adult↗

PubMed1996

[Pneumocystis carinii lung infections in AIDS patients: a study with high-resolution computed tomography (HRCT)].

In 1993, a hundred and fifty AIDS patients were submitted to high-resolution CT (HRCT). In 102 patients, bronchoalveolar lavage and/or transbronchial biopsy findings suggested the diagnosis of Pneumocystis carinii pneumonia--a pure Pneumocystis carinii infection in 75 patients and associated with other pathogenic agents in 27. We report the most common HRCT patterns, such as ground-glass opacities, cysts, interstitial changes and nodules. Ground-glass opacities were demonstrated in 57.8% of cases, cysts in 44.1%, interstitial involvement in 52.9% and nodules in 28.4%. HRCT permitted lung disease to be demonstrated in 55% of our patients, suffering from impaired breathing, with negative chest films. Respiratory function tests and gallium scintigraphy show their low specificity in the diagnosis of Pneumocystis carinii infection because, although depicting diffuse interstitial involvement, they fail to detect the pathogenic agent. As for hemogasanalysis, in the presence of hypoxia, this technique can suggest the diagnosis of Pneumocystis carinii infection, while the pathogenic agent can be isolated with bronchoalveolar lavage, which demonstrates the simultaneous decrease in CD4 and increase in CD8 lymphocytes, respectively. To conclude, HRCT does detect the basic changes occurring in Pneumocystis carinii pneumonia, thus contributing to the diagnosis of this condition.

AIDS-Related Opportunistic Infections↗