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PubMed1987

[Detection of pathogens in bacterial meningitis. Comparison of Gram staining, culture, latex agglutination test and counterimmunoelectrophoresis].

To look for the etiologic agent in cerebrospinal fluids of 44 children with meningitis, Gram stain, traditional culture, latex agglutination and counterimmunoelectrophoresis were simultaneously performed. The sensitivity of all four methods was roughly equal. The outstanding importance of the Gram technique has to be emphasized, however. Differences with regard to antigen detection could be observed in patients previously treated with antibiotics, where etiologic agents could still be detected by latex agglutination and counterimmunoelectrophoresis, but not by traditional cultures. A further advantage of the two methods is that they provide results rapidly. The use of all four methods simultaneously allows for optimal results in the detection of causative organisms in cerebrospinal fluid in meningitis.

Bacteria↗
PubMed2026

Project ODIN: advancing environmental genomic surveillance for public health across sub-Saharan Africa.

Persistent SARS-CoV-2 transmission, ongoing mpox outbreaks, and the continued spread of endemic diseases such as typhoid fever and cholera underscore the urgent need for global, multiomics surveillance. In this Personal View, we present Project ODIN, a consortium of European and African partners launched in 2023 that aims to meet this challenge by deploying innovative systems for near real-time pathogen detection and actionable public health insights. The project is a collaboration between high-income and low-income countries in northern Europe and sub-Saharan Africa. Focusing on low-income and middle-income countries, ODIN integrates metagenomics with mobile laboratory systems for comprehensive pathogen monitoring across diverse environments. ODIN emphasises standardised sampling, bioinformatics pipelines, and data-sharing protocols to ensure reliable, interoperable results while addressing infrastructure and resource limitations. By bridging gaps in genomic surveillance, these initiatives seek to strengthen outbreak preparedness, improve pathogen detection, monitor antimicrobial resistance, and provide a holistic approach to One Health challenges. Together, these innovations could advance global surveillance capacity-particularly in under-resourced regions-paving the way for effective disease control and evidence-based policy making.

Humans↗
PubMed1986

Detection of pathogenic Campylobacter species in blood culture systems.

Because differences in recognition of Campylobacter fetus and Campylobacter jejuni in systemic infections may be due partially to differences in the ability to cultivate these organisms, we studied their growth characteristics in two widely used blood culture systems. In the Roche Septi-Chek system (Hoffman-La Roche, Inc., Nutley, N.J.), over a broad range of inocula all strains were detected in broth within 2 days and on paddles within 3 days. In the BACTEC 6B aerobic bottles (Johnston Laboratories, Inc., Towson, Md.), C. jejuni and C. fetus took a median of 5 and 3 days, respectively, to reach the growth index threshold. However, in the BACTEC 7D anaerobic bottles, C. fetus required a median of 2 days to reach the growth index threshold, whereas for C. jejuni the median was greater than 10 days. The poor performance of C. jejuni in both BACTEC systems may have been due to unfavorable incubation atmospheres and may partially explain why C. jejuni bacteremia is so infrequently detected. Overall, the Roche Septi-Chek system was excellent for detecting Campylobacter strains in blood cultures.

Aerobiosis↗
PubMed2003

Rapid detection of pathogenic fungi from clinical specimens using LightCycler real-time fluorescence PCR.

In the study presented here a LightCycler real-time PCR system was used for the diagnosis of fungal infections from clinical tissue samples. Nine specimens were investigated from six patients with suspected or proven invasive fungal infections. Seven of nine samples were positive in a broad-range fungal PCR assay. In four samples, Aspergillus fumigatus was detected both by a species-specific hybridization assay as well as by sequencing of amplification products. In addition, the broad-range fungal PCR assay and PCR sequencing detected and identified, respectively, the following organisms in the specimens noted: Candida albicans in a culture-negative liver biopsy, Histoplasma capsulatum in a bone marrow sample, and Conidiobolus coronatus in a facial soft tissue specimen. Real-time PCR is a promising tool for the diagnosis of invasive fungal infections in human tissue samples and offers some advantages over culture methods, such as rapid analysis and increased sensitivity.

Aspergillus fumigatus↗
PubMed2005

Molecular markers for detection of pathogenic Escherichia coli strains belonging to serogroups O 138 and O 139.

Escherichia coli strains belonging to O-serogroup 138 and 139 are important as disease agents in pigs causing post-weaning diarrhea and edema disease. Several types of shiga toxin-producing O 138 and O 139 strains were isolated from diarrheic humans and from cattle and food of bovine origin. Serotyping is the current method for detection of O 138 and O 139 strains but its applicability can be limited due to the presence of capsules and capsular-like bacterial surface antigens and in the case of rough LPS. To overcome these difficulties for diagnosis, we have developed a specific PCR method suitable for detection of different types of O 138 and O 139 strains. The O-antigen gene clusters of E. coli O 138 and O 139 type strains were sequenced, and the genes were identified on the basis of homology. By screening against 186 E. coli and Shigella type strains, two genes specific to each of E. coli O 138 and O 139 were identified, respectively, and were tested on 15 clinical and environmental isolates of those two serogroups in a double-blind test. The sensitivity of the PCR assays was determined, and the detection limits were 2 pg per mul of chromosomal DNA and 2 CFU per 10 g of water or pork samples. PCR-based detection of O-antigen specific genes of E. coli O 138 and O 139 was shown to be accurate, highly sensitive and rapid, and is suggested as a new diagnostic tool for investigations of infections and outbreaks with these strains in animals and humans and for control of food.

Animals↗
PubMed1994

[Empirical antimicrobial therapy in neutropenic patients. Results of a multicenter study by the Infections in Hematology Study Group of the Paul Ehrlich Society].

BACKGROUND: Severe infections are the predominant cause of treatment failure in patients with high grade malignant hematological disorders undergoing intensive chemotherapy. PATIENTS AND METHODS: In a multicenter trial of the Paul Ehrlich Society (PEG) study group, febrile neutropenic patients with acute leukemias or other high grade hematological malignancies were randomized for a three phase sequential antimicrobial intervention comparing different widely applied regimes for empirical therapy. Patients with clinically documented infections were treated according to a modification depending on the respective source of infection, whereas in patients with microbiologically documented infections, treatment could be adapted to the sesceptibility patterns of detected pathogens. Criteria for evaluation as well as time points for response assessment and treatment escalation were strictly prescribed by the study protocol. RESULTS: Of 1573 evaluable patients, 50.9% had fever of unknown origin (FUO) throughout the study period, 17.1% had lung infiltrates, 14.1% primary bacteremia or fungemia (B/F), 12.6% other clinically documented (CDI) and 5.3% other clinically as well as microbiologically documented infections (CMDI). Cumulative response rate (CR) in patients with FUO was 91.3%, a significant difference between various regimens could not be detected in either of the three treatment phases. Patients with lung infiltrates had a significantly worse treatment outcome as compared to patients with other documented infections or with FUO (61.3% vs 82.9% vs 91.3%). Gram-positive pathogens dominated in case of microbiologically documented infections (MDI), whereas the proportion of fungal infections increased dramatically in MDI with pathogens detected only after more than six days under study. Of numerous prognostic factors analyzed, only the trend in white blood cell counts had a significant impact on treatment outcome. CONCLUSION: Infection-related mortality in neutropenic patients with high grade hematological malignancies can be markedly reduced by a systematically escalating interventional antimicrobial therapy. Early systemic antifungal treatment, especially in patients with lung infiltrates, might further improve treatment results.

Adolescent↗
PubMed2002

Development of a nested PCR assay to detect the pathogenic free-living amoeba Naegleria fowleri.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a fatal disease of the central nervous system that is acquired while swimming or diving in freshwater. A cDNA clone designated Mp2C15 obtained from N. fowleri was used as a probe to distinguish N. fowleri from other free-living amoebae. The Mp2C15 probe hybridized to genomic DNA from pathogenic N. fowleri and antigenically related non-pathogenic N. lovaniensis. Mp2C15 was digested with the restriction enzyme XbaI, resulting in two fragments, Mp2C15.G and Mp2C15.P. Four species of Naegleria and four species of Acanthamoeba were examined for reactivity with Mp2C15.P. Mp2C15.P was specific for N. fowleri and was used in the development of a nested PCR assay which is capable of detecting as little as 5 pg of N. fowleri DNA or five intact N. fowleri amoebae. In summary, a rapid, sensitive, and specific assay for the detection of N. fowleri was developed.

Amebiasis↗
PubMed2005

[Study on the frequency of antibiotics use per day among inpatients in 151 hospitals in 2003].

OBJECTIVE: To investigate the antibiotics use in inpatients with hospitals under different scales. METHODS: On the day of August 7, 2003, antibiotics use among inpatients were investigated, medical order to each inpatient was checked and forms were filled. RESULTS: Data showed that 54.86% of the 89,539 inpatients from 151 hospitals were using antibiotics, out of which 48.98% were used for therapeutic purposes while 35.44% for prophylaxis, and 15.58% for both therapy and prophylaxis. Among those who had received antibiotics, 61.43% received one, 33.65% received two and 4.92% received 3 or more agents. In patients who were on antibiotics for therapy, only 23.92% of the samples were sent for pathogens detection. The prevalence of antibiotics use was different among different hospitals, with teaching hospitals having the lowest and hospitals with less than 300 beds were higher than those with more than 300 beds. Departments of respiratory diseases, intensive care unit, and pediatrics had the higher use of antibiotics. CONCLUSION: Inpatients in 151 hospitals had high prevalence of antibiotics use, especially for prophylaxis use but pathogen detection was seldomly done. It is necessary to strengthen the rational use and management of antibiotics use in the hospitals.

Anti-Bacterial Agents↗
PubMed1990

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water.

Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach. Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes. Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E. coli and enteric pathogens Salmonella and Shigella. Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens.

Bacteriological Techniques↗
PubMed2001

Glutamate decarboxylase genes as a prescreening marker for detection of pathogenic Escherichia coli groups.

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx(1) and stx(2) was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.

Biomarkers↗
PubMed2026

Molecular epidemiology and phylogeographic architecture of oncogenic intracellular bacteria in cervical cancer patients across Northern China.

BACKGROUND: Oncogenic intracellular bacteria, including Chlamydia trachomatis, Mycoplasma genitalium, and Fusobacterium nucleatum, have emerged as significant contributors to cervical carcinogenesis. Despite growing interest in microbial oncology, the molecular epidemiological landscape and phylogeographic distribution of these pathogens in Northern China remain poorly characterized. This study aimed to determine the prevalence, co-infection patterns, genotypic diversity, and spatial phylogeographic clustering of oncogenic intracellular bacteria among cervical cancer patients across five provinces of Northern China. METHODS: A cross-sectional, multi-center study was conducted between March 2022 and November 2024 across Shaanxi, Heilongjiang, Beijing, Shandong, and Inner Mongolia. Cervical swab specimens were collected from 1247 confirmed cervical cancer patients. Pathogen detection was performed using multiplex real-time polymerase chain reaction, 16S rRNA gene amplicon sequencing, and whole-genome sequencing. Phylogeographic analyses employed maximum likelihood and Bayesian evolutionary inference frameworks. Statistical analyses included multivariate logistic regression and geographic information system-based spatial clustering. RESULTS: The overall prevalence of at least one oncogenic intracellular bacterium was 68.3% (n&#xa0;=&#xa0;852). Chlamydia trachomatis was the most prevalent pathogen detected in 41.2% of participants. Co-infection with two or more bacteria was identified in 29.7% of cases and was independently associated with advanced-stage cervical cancer (adjusted odds ratio&#xa0;=&#xa0;2.87; 95% confidence interval: 1.94 to 4.23; p&#xa0;<&#xa0;0.001). Phylogeographic analysis revealed three distinct molecular clades with evidence of bidirectional gene flow between Shaanxi and Heilongjiang. Whole-genome sequencing identified 14 novel virulence gene variants not previously characterized in Chinese clinical isolates. CONCLUSIONS: Oncogenic intracellular bacteria are highly prevalent and genotypically diverse among cervical cancer patients in Northern China. The identified phylogeographic clustering and novel virulence variants have direct implications for regional screening programs, targeted antimicrobial strategies, and the development of region-specific molecular diagnostic panels.

Cervical cancer↗
PubMed2005

Detection and pathogenicity of human metapneumovirus respiratory infection in pediatric Italian patients during a winter--spring season.

BACKGROUND: Some diagnostic, epidemiological and clinical features of the recently discovered human metapneumovirus remain to be investigated. OBJECTIVES: To study the best approach for the diagnosis of human metapneumovirus infections by both conventional and molecular methods, along with the human metapneumovirus circulation rate in northern Italy and the severity of human metapneumovirus respiratory infections in a pediatric patient population. STUDY DESIGN: Nasopharyngeal aspirates (NPA) were taken from 306 pediatric patients during the winter-spring season 2003-2004, and examined for conventional respiratory viruses by direct fluorescent staining and cell culture, while human coronavirus and human metapneumovirus were sought by RT-PCR. RESULTS: RT-PCR detected human metapneumovirus in 40/306 (13.1%) children positive for respiratory viruses, with an incidence intermediate between that of respiratory syncytial virus (58 patients, 18.9%) and that of influenzavirus infections (29 patients, 9.5%). Phylogenetic analysis showed cocirculation of both human metapneumovirus types (A and B) as well as their relevant subtypes (A1-A2 and B1-B2). Clinically, human metapneumovirus was found to be second to human respiratory syncytial virus alone, as a cause of respiratory tract infections, while duration of virus excretion appeared to correlate with severity of infection, and virus load in NPA with the stage of respiratory infection. CONCLUSION: (i) Human metapneumovirus is a major viral pathogen in the Italian pediatric patient population; (ii) the severity of lower respiratory tract infections approaches that of human respiratory syncytial virus; (iii) there are preliminary indications that the duration of virus excretion may reach 2-3 weeks and that the level of viral load in NPA correlates with the clinical stage of human metapneumovirus infection.

Animals↗
PubMed2003

An applications-focused review of comparative genomics tools: capabilities, limitations and future challenges.

A team at the Lawrence Livermore National Laboratory (LLNL) was given the task of using computational tools to speed up the development of DNA diagnostics for pathogen detection. This work will be described in another paper in this issue (see pages 133-149). To achieve this goal it was necessary to understand the merits and limitations of the various available comparative genomics tools. A review of some recent tools for multisequence/genome alignment and substring comparison is presented, within the general framework of applicability to a large-scale application. We note that genome alignments are important for many things, only one of which is pathogen detection. Understanding gene function, gene regulation, gene networks, phylogenetic studies and other aspects of evolution all depend on accurate nucleic acid and protein sequence alignment. Selecting appropriate tools can make a large difference in the quality of results obtained and the effort required.

Algorithms↗
PubMed1993

[The effect of cefaclor on the nasopharyngeal pathogens in children].

Nasopharyngeal specimens were taken from 29 children with acute otitis media, 14 children with chronic sinusitis at acute exacerbation and 2 children with streptococcal pharyngitis who had received cefaclor for 14 days. The study was designed to compare the microbiologic flora of the nasopharynx before the treatment of these diseases with that after the treatment. In this report the subjects were limited to the children who had not received antibiotics within 1 month. Pathogens detected on initial examination were 49 strains consisting of 40 strains of H. influenzae, 3 strains of S. aureus, 5 strains of S. pyogenes, and 1 strain of S. pneumoniae. Two strains were detected in 4 patients. Pathogens remained after treatment in 29 patient, with an unchanged number of pathogens in 12 patients, a decreased number in 14, and microbial substitution in 3. All of the remaining pathogens were H. influenzae, and minimum inhibitory concentration (MIC) changed in 5 of the 26 patients in whom H. influenzae was detected before and after treatment. In a patients with increased MIC, the strain changed from a sensitive strain with an MIC of lower than 3.13 micrograms/ml to a highly resistant strain with an MIC of 25.0 micrograms/ml or higher. By contrast, in 2 patients with decreased MIC, the strain changed from a highly resistant strain with an MIC of 12.5 micrograms/ml or higher to a sensitive strain with an MIC of lower than 3.13 micrograms/ml. In the other patient, MIC decreased to 3.13 micrograms/ml after it had increased from 3.13 micrograms/ml to 25.0 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2003

Analysis of environmental Escherichia coli isolates for virulence genes using the TaqMan PCR system.

AIMS: To assess the presence of virulence genes in environmental and foodborne Escherichia coli isolates using the TaqMan PCR system. METHODS AND RESULTS: Three TaqMan pathogen detection kits called O157:H7, StxI and StxII were used to investigate the presence of virulence genes in Escherichia coli isolates. All 54 foodborne E. coli O157:H7 isolates showed expected results using these kits. Ninety (15%) of 604 environmental isolates gave positive amplification with an O157:H7-specific kit. TaqMan PCR amplification products from these 90 isolates were analysed by agarose gel electrophoresis, and 90% (81 of 90) of the environmental samples contained the expected PCR product. Sixty-six of these 90 were chosen for serotyping tests and only 35% (23 of 66) showed agglutination with both anti-O157 and anti-H7 antibodies. Further ribotyping of 16 sero-positive isolates in an automated Riboprinter did not identify these to be O157:H7. Multiplex PCR with primers for eaeA, stxI and stxII genes was used to confirm the TaqMan results in 10 selected environmental isolates. CONCLUSIONS: All three TaqMan pathogen detection kits were useful for virulence gene analysis of prescreened foodborne O157:H7 isolates, while the O157:H7-specific kit may not be suitable for virulence gene analysis of environmental E. coli isolates, because of high false positive identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to rapidly identify the presence of pathogenic E. coli in food or environmental samples is essential to avert outbreaks. These results are of importance to microbiologists seeking to use TaqMan PCR to rapidly identify pathogenic E. coli in environmental samples. Furthermore, serotyping may not be a reliable method for identification of O157:H7 strains.

Bacterial Typing Techniques↗