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Genomic approaches to the study of the expression of plant genes induced in response to disease and attack are now showing that there is an intimate association between pathogen perception and general stress detection.
An array of PCR microchips for rapid, parallel testing of samples for pathogenic microbes is described. The instrument, called the Advanced Nucleic Acid Analyzer (ANAA), utilizes 10 silicon reaction chambers with thin-film resistive heaters and solid-state optics. Features of the system include efficient heating and real-time monitoring, low power requirements for battery operation, and no moving parts for reliability and ruggedness. We analyzed cultures of Erwinia herbicola vegetative cells, Bacillus subtilis spores, and MS2 virions, which simulated pathogenic microbes such as Yersinia pestis, Bacillus anthracis spores, and Venezuelan equine encephalitis, respectively. Detection of microbes was achieved in as little as 16 min with detection limits of 10(5)-10(7) organisms/L (10(2)-10(4) organisms/mL).
Recent reports have suggested that examination of one stool specimen is sufficient to detect protozoa in symptomatic patients. To verify this hypothesis, we examined specimens submitted to the regional laboratory of a large, prepaid health plan that provides primary care to a membership representative of the general population in northern California. We examined the number and results of stool specimen examinations in the two-month period before detection of Entamoeba histolytica in 125 infected persons, of Giardia lamblia in 154 infected persons, and of Dientamoeba fragilis in 60 persons infected with this parasite. About 13% of the entire group had a single stool specimen examined, 19% had two examined, 65% had three examined, and 6% had more than three examined. Most infections were detected in the first specimen or specimens submitted, but many were not detected until later examination. Thirteen patients with E. histolytica (10.4%) required 4-9 examinations. The sensitivity of one examination was compared with that of three examinations; with the additional examinations, the yield increased 22.7% (95% confidence interval [CI] = 11.8-33.5%) for E. histolytica, 11.3% (95% CI = 6.9-81.8%) for G. lamblia, and 31.1% (95% CI = 17.7-54.4%) for D. fragilis. Our results suggest that a single stool specimen examination will miss many pathogenic protozoan infections in symptomatic persons.
SUMMARY: nf-UnO is a pipeline implemented in Nextflow to identify novel pathogens from metagenomic shotgun sequencing of epidemiologically related foodborne outbreak specimens. nf-UnO uses MIDAS2, metagenomic co-assembly, multiple binning programs, and read mapping to metagenomically assembled genomes to detect potential etiological agents found in common across outbreak specimens. AVAILABILITY AND IMPLEMENTATION: https://github.com/uel3/nf-UnO.
Sepsis is a severe and life-threatening disease that requires a rapid and reliable diagnosis. The detection of microorganisms in blood culture and the results of in vitro anti-microbial susceptibility testing are prerequisites for a directed and cost-effective antimicrobial therapy. Microbiological results are difficult to interpret if contamination of the blood culture with colonizing or environmental germs cannot be ruled out. The source of contamination is predominantly inappropriate withdrawal of blood. Occasionally, contamination of blood cultures occurs in the laboratory. A potentially relevant blood culture result is already indicated by the isolated pathogen itself. In addition, a short time between the collection of blood and the isolation of the microorganism as well as the detection of the same pathogen in several blood cultures even more suggest a clinically relevant result.
OBJECTIVE: The aim of the study was to examine the prevalence of selected periodontal pathogens associated with HIV and non-HIV related periodontal lesions. METHODS: Subgingival plaque samples were obtained from both HIV-seropositive and HIV-seronegative patients affected with periodontal disease. DNA probes were used to detect Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Eikenella corrodens and Campylobacter rectus. RESULTS: A actinomycetemcomitans, P. intermedia and B. forsythus (P < 0.05) were more prevalent in HIV-seronegative patients with rapidly progressive periodontitis. Only C. rectus was slightly more prevalent in HIV-seropositive subjects with periodontal diseases, but this was not significant.
Vibrio parahaemolyticus has been detected in the Natal and Eastern Cape costal water of South Africa. As a proven cause of gastro-enteritis or a potential pathogen to humans, it is an organism of importance to public health. Outlines of the history, pathogenicity and identification are given, as well as the results of the local investigation of this organism.
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A microattachment assay for quantitating adherence of radiolabelled Mycoplasma pneumoniae to human WiDr cell culture monolayers is described. Preincubating the WiDr cell monolayers with a protein-rich extract of M. pneumoniae inhibited the subsequent attachment of radiolabelled organisms. Competitive attachment inhibition provided a quantitative procedure to determine M. pneumoniae-binding components in the extract. The microattachment assays also measured attachment inhibition by the sialoglycoconjugates ceruloplasmin, orosomucoid and gangliosides, indicating that these reagents may be structural analogues of the mammalian cell receptor. Attachment of virulent M. pneumoniae strains to glutaraldehyde-treated monolayers was reduced approximately 60% and showed a different temperature dependence compared with untreated cells. These results suggest that maximal attachment of virulent M. pneumoniae may require two or more different receptors and binding components.
The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes. Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA. Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack. The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes. PCR reactions using SYBR Green dye allowed detection of E. coli ATCC 11775 and E. coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present. DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes. Positive results were observed at 32 and 22 min of assay time, respectively. A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time. Background fluorescence associated with the use of the probe was negligible. The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002.
Between 1976 and 1986 the secretions from the pouch of Douglas of 1,219 patients with non-inflammatory genital diseases were microbiologically studied. Microorganisms were detected in 3.4%. In the majority of cases they were physiological skin organisms, with a count of less than 10 per ml of Douglas fluid. These findings support the view, that secondary contamination of the specimens occurred during removal, transportation, or laboratory processing.
When a DNA probe hybridizes a DNA target and generates a G/A mismatch in the probe-target DNA heteroduplex, the mismatch enzyme, mutY, will cut the A base at the site of the mismatch. This specific cleavage at the mismatched A on the known probe will reveal the complementary DNA sequences of the targets. This study shows mismatch cleavage assays identify the complementary sequence of cryptic plasmid target in the extract of chlamydia infected cells in culture. In addition, the specific cleavage at a single base permits differentiation of two sequences with one base difference. This was shown in differentiating the subtypes of human immunodeficiency virus (HIV) type 1. The addition of amines in the assays increases the sensitivity by freeing the target for recycling. The combined assay system of high sensitivity and demonstrated specificity allows further evaluation for direct identification of pathogenic microorganisms in patient samples.
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ATP is a kind of necessary metabolites in the living cells. We detected ATP contents in 222 bacteriuria or the urine germ by bioluminescence method. The results showed that the patient had been infected when the mean value was > 130.9 +/- 11.38 and had not when the mean value was < 32.9 +/- 2.26. The results were the same with the Gram Stain, L-form bacteriuria culture and the common bacteriuria culture. The mean values of positive specimens are higher than that of the negative specimens by the bioluminescence. Thus, it is indicated that the bioluminescence method has high sensitivity. This method can be used to detect L-form bacteriuria in the urine germ quickly and is important for early diagnosis by the doctors.
The prevalence of antibodies against Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, was determined in a group of blood donors from the Würzburg area (Southern Germany). 26 of 472 donors (5.5%) tested positive in a hemagglutination test. When performing immunoblots only 13 donors (2.7%) gave rise to B. burgdorferi-specific antibodies. 9 of them were examined in more detail by anamnesis, physical examination, determination of inflammation parameters of the blood and polymerase chain reaction (PCR) analysis of urine. All persons were asymptomatic for Lyme borreliosis. One of 4, who remembered a tick bite, actually had suffered from an erythema migrans 5 years ago. Another one had been affected by fever, headaches and pains in the limbs, arthralgia and motoric disorder in both hands 6 months before examination. Analysis of the blood did not provide any evidence of an acute infection. In the urine of 2 donors we detected B. burgdorferi-specific DNA by PCR. No seroconversion due to blood transfusion could be observed, when 9 recipients of blood products provided by the 13 seropositive donors were serologically reexamined. PCR analysis of urine samples of 5 recipients was also negative.
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Dark-field microscopy, fuchsin stain and Fontana silver stain were used for a rapid detection of spirochaetes in fresh materials from patients with Lyme-borreliosis. The spirochaetes were shown to be present in 2 cerebrospinal fluids, 2 synovial fluids and in one brain cyst fluid. It is pointed out that these techniques require further improvement and optimization.