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PubMed2001

Incidence of respiratory syncytial virus-positive hospitalizations in Germany.

Epidemiological data, especially population-based data, on respiratory syncytial virus (RSV)-related hospitalizations in Germany have been lacking to date. Since Palivizumab (Synagis, Abbott, USA) is already available and new vaccines for active immunization are under development, these data are urgently needed. From July 1996 to June 1999, nasopharyngeal aspirates of children hospitalized in Kiel with an acute respiratory tract infection were tested by multiplex reverse transcriptase polymerase chain reaction. Of 1,241 patients, 150 (12.1%) were RSV positive. RSV was the predominant pathogen detected through the end of the second year. In 37 (25%) children an underlying condition was present. For the city of Kiel and close surroundings, the cumulative incidence of RSV-positive hospitalizations in infants was 1,214/10(5) (725/10(5) in children less than 2 years). For those children less than 2 years old born with a gestational age of less than 32 weeks or 32-37 weeks, the cumulative incidence was 2,025/10(5) and 1,202/10(5), respectively (dose-effect response). For the group less than 32 weeks of age, bronchopulmonary dysplasia (BPD) as an underlying condition carried a relative risk of 17.8. The RSV season began between the end of September and January and ended between March and July. The population-based incidence of RSV-positive hospitalizations in Kiel is close to that reported from the UK and Scandinavia. Throughout Germany, approximately 10.000 RSV-related hospitalizations in infants can be expected annually. Prematurity is an effect modifier and BPD a strong risk factor for RSV-positive hospitalization in population-based studies. There is considerable variation in the start and end of the yearly epidemic.

Acute Disease↗

PubMed2026

CRISPR-Cas and Infectious Diseases: A Decade of Translational Advances in Molecular Biotechnology.

CRISPR-Cas systems have emerged as a versatile tool for diagnosing, treating, and preventing infectious diseases. This review highlights translational advancements in CRISPR-Cas-based applications, concentrating on the past decades in diagnostics, therapeutic genome editing, and vaccine development. The article highlights key platforms like DETECTR and SHERLOCK, which enable rapid, sensitive pathogen detection, and explores CRISPR-Cas9 systems in therapeutic strategies for directly targeting viral genomes and combating antimicrobial resistance. It also examines the role of CRISPR-Cas9 in engineering live-attenuated and personalized neoantigen vaccines. Principal findings demonstrate a clear progression from experimental proof-of-concept to preclinical applications primarily in CRISPR-based diagnostics and the engineering of live-attenuated vaccine candidates, whereas translation in CRISPR-based therapeutics and personalized neoantigen vaccines for infectious diseases remains at earlier, more exploratory stages. CRISPR-based diagnostics have progressed further toward clinical evaluation than therapeutics due to delivery and safety constraints, while personalized neoantigen vaccines are included mainly as an emerging, comparative concept for infectious diseases rather than a mature application. This review uniquely integrates CRISPR-based diagnostics, therapeutics, and vaccine development within a single infectious disease framework, critically assesses their current maturity, and systematically highlights technical, regulatory, and ethical barriers alongside realistic future priorities. The review concludes that while CRISPR-Cas holds transformative potential for infectious disease management, significant challenges in delivery efficiency, off-target effects, and ethical regulation must be addressed to ensure safe and equitable clinical translation.

Humans↗

PubMed1989

The polymerase chain reaction.

The polymerase chain reaction (PCR) is a novel technique that amplifies specific sequences with remarkable efficiency. Repeated cycles of denaturation, primer annealling and extension carried out with the heat stable enzyme, Taq polymerase, leads to exponential increases in the target DNA sequences. The technique has been widely applied in immunology to define HLA polymorphism, T-cell receptor and immunoglobulin diversity, pathogen detection, quantification of lymphokines and lymphoma or leukaemia detection. In addition, it has been very widely applied in all areas of molecular biology and human genetics. Because of its simplicity and sensitivity, PCR represents a major technical development for molecular biologists and will continue to have a great impact on molecular immunology. This review by John Bell describes the basic PCR technique and some variations on it and briefly explores their uses in molecular immunology.

DNA↗

PubMed1996

Microbiological analysis of 'black pudding', a Trinidadian delicacy and health risk to consumers.

The prevalences of Salmonella, Escherichia coli and Staphylococcus aureus in black pudding which originated from local vendors and supermarkets in Trinidad were determined. The enterotoxigenicity of S. aureus strains and occurrence of O157:H7 strains amongst E. coli isolates were also investigated. For the 100 black puddings each sampled from supermarkets and vendors, the mean total aerobic plate count (TAPC) per g was 1.8 x 10(7) +/- 1.5 x 10(7) and 1.5 x 10(8) +/- 2.3 x 10(8), respectively. E. coli was isolated from 56 (56.0%) black pudding samples from supermarkets with a mean count per g of 9.2 x 10(6) +/- 7.9 x 10(6) compared to a prevalence of 79% (79 of 100) and mean count per g of 3.2 x 10(7) +/- 4.7 x 10(7) for samples from local vendors. The difference between prevalences was statistically significant (P < or = 0.001; chi 2). Only 1 (2.2%) of 45 strains of E. coli from supermarket-purchased pudding tested, was an 0157:H7 strain compared to 9 (13.6%) of 66 strains of E. coli from vendor-sold black pudding. The difference was not statistically significant (P > or = 0.05; chi 2). Five (5.0%) of 100 black pudding samples from supermarkets yielded Salmonella, with S. ohio being the predominant serotype. For vendor-sold black pudding however, 11 (11.0%) samples were positive for Salmonella with a new serotype, S. unnamed (4,12:d-) being responsible for 50% (6 of 12) of isolates from this source. Forty samples each of black pudding from supermarkets and vendors were all (100.0%) positive for S. aureus with mean counts per g being 3.1 x 10(5) +/- 8.8 x 10(5) and 3.3 x 10(6) +/- 7.7 x 10(6), respectively. Overall, 27 (33.8%) of 80 strains of S. aureus tested were enterotoxigenic producing staphylococcal enterotoxins A(SEA), SEB, SEC, SED or a combination. It was concluded that black pudding poses a high risk to consumers based on the prevalence, microbial load and toxigenicity of the pathogens detected.

Culture Media↗

PubMed1988

Detection of toxigenic Escherichia coli using biotin-labelled DNA probes following enzymatic amplification of the heat labile toxin gene.

Several types of DNA probes labelled with biotin were compared for their sensitivity to detect the heat labile toxin (LT) gene in toxigenic Escherichia coli. In addition, a procedure was developed for enzymatically amplifying LT gene sequences in toxigenic E. coli. Probes were labelled with biotinylated nucleotides by either nick translation; 3' tailing; primer extension of probe DNA cloned into bacteriophage M13; sandwich hybridization; or oligolabelling of isolated DNA fragments. A single stranded probe consisting of a DNA fragment from the LT gene cloned into the bacteriophage M13mp18 and detected by hybridization to oligolabelled biotinylated M13mp18 RF DNA in a sandwich hybridization was able to detect as little as 10 pg of toxin gene DNA. Cloned LT gene DNA was serially diluted and amplified enzymatically using synthetic oligonucleotide primers. Amplified DNA was detected using biotin-labelled M13-based probes. As little as 1 fg of LT DNA could be amplified to detectable levels by this method. Experiments with LT+ bacteria resulted in the detection of as few as 1000 bacteria. The combination of enzymatic amplification coupled with M13-based DNA probes provides a highly sensitive tool for detecting pathogenic microorganisms.

Bacterial Toxins↗

PubMed2006

Microarray technology in obstetrics and gynecology: a guide for clinicians.

Microarrays can be constructed with dozens to millions of probes on their surface to allow high-throughput analyses of many biologic processes to be performed simultaneously on the same sample. Microarrays are now widely used for gene expression analysis, deoxyribonucleic acid resequencing, single-nucleotide polymorphism genotyping, and comparative genomic hybridization. Microarray technology is accelerating research in many fields and now microarrays are moving into clinical application. This review discusses the emerging role of microarrays in molecular diagnostics, pathogen detection, oncology, and pharmacogenomics.

Chromosome Mapping↗

PubMed2005

A high density microelectrode array biosensor for detection of E. coli O157:H7.

A high density microelectrode array biosensor was developed for the detection of Escherichia coli O157:H7. The biosensor was fabricated from (100) silicon with a 2 microm layer of thermal oxide as an insulating layer, an active area of 9.6 mm2 and consists of an interdigitated gold electrode array. The sensor surface was functionalised for bacterial detection using heterobifunctional crosslinkers and immobilised polyclonal antibodies to create a biological sensing surface. Bacteria suspended in solution became attached to the immobilised antibodies when the biosensor was tested in liquid samples. The change in impedance caused by the bacteria was measured over a frequency range of 100 Hz-10 M Hz. The biosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The biosensor was able to discriminate between cellular concentrations of 10(4)-10(7)CFU/mL and has applications in detecting pathogens in food samples.

Antibodies↗

PubMed2005

Optical fiber-based sensors: application to chemical biology.

Optical fibers have been used to develop sensors based on nucleic acids and cells. Sensors employing DNA probes have been developed for various genomics applications and microbial pathogen detection. Live cell-based sensors have enabled the monitoring of environmental toxins, and have been used for fundamental studies on populations of individual cells. Both single-core optical fiber sensors and optical fiber sensor arrays have been used for sensing based on nucleic acids and live cells.

Biosensing Techniques↗

PubMed2026

Tn125-borne blaNDM-1 is decoupled from clonal background in a transcontinental Acinetobacter baumannii ST126/KL14 lineage.

BACKGROUND/OBJECTIVES: Carbapenem-resistant A. baumannii (CRAB) is a WHO Critical Priority pathogen. The blaNDM-1-carrying ST126/KL14 lineage has been independently reported from Vietnam (2015), Malaysia (2016), the USA (2023-2026), and Costa Rica (2024). Whether these geographically distinct reports represent a single transcontinental clone and through what mechanism blaNDM-1 disseminates has not been formally tested. METHODS: We performed comprehensive whole-genome reanalysis of the Vietnamese sentinel isolate DMS06669_L1 using three nested panels (n = 19, n = 138, and n = 609 Vietnamese A. baumannii genomes) and surveyed 429 plasmids extracted from 99 NDM-1 A. baumannii genomes retrieved from NCBI Pathogen Detection. RESULTS: Four ST126/KL14 isolates share high inter-regional average nucleotide identity (ANI; 99.77-99.92%) but wide intra-clade core-SNP distances (41-731 SNPs, well above the ∼20-40-SNP range typical of single-outbreak transmission clusters) and lack a significant molecular clock, consistent with a related transcontinental lineage rather than a single recent clone. NDM-1 plasmid evolution is statistically uncorrelated with chromosomal sequence type (Spearman ρ = 0.131, P = 0.573). At 609-strain population scale, under a fragmentation-aware detection criterion, all 41 blaNDM-1-carrying Vietnamese strains also carry ISAba125, with none carrying blaNDM-1 without it (Fisher exact test; Haldane-Anscombe-corrected OR ≈2.0 × 10³, 95% CI 1.2 × 10² to 3.4 × 10⁴; P = 4.74×10⁻⁴⁸; φ = 0.79). CONCLUSIONS: The blaNDM-1 dissemination pattern in this ST126/KL14 lineage is primarily consistent with Tn125 transposition acting alongside plasmid-borne spread. Standard MLST-based surveillance is insufficient; multi-level genomic monitoring - including chromosomal and plasmid-level detection of the Tn125/ISAba125 unit - is required to track this resistance threat.

A. baumannii↗

PubMed2024

Offering complex genomic screening in acute pediatric settings: Family decision-making and outcomes.

PURPOSE: Families of children in pediatric acute care who are offered ultrarapid genomic sequencing are making complex decisions during a high-stress period. To reduce complexity for families and clinicians, we offered genomic screening for the child and parents after the completion of diagnostic testing. We evaluated uptake, understanding, and service delivery preferences. METHODS: A cohort of 235 families who had completed ultrarapid diagnostic genomic sequencing at 17 Australian hospitals were offered up to 3 screens on their genomic data: pediatric-onset, adult-onset, and expanded couple carrier screening. We investigated decision making, understanding, and service delivery preferences using surveys at 3 time points (pre counseling, post counseling, and post result) and performed inductive content analysis of pretest genetic counseling transcripts. RESULTS: A total of 119 families (51%) attended genetic counseling with 115 (49%) accepting genomic screening. Survey respondents were more likely to find decisions about couple carrier screening easy (87%) compared with adult (68%; P = .002) or pediatric (71%; P = .01) screening decisions. All respondents with newly detected pathogenic variants accurately recalled this 1 month later. A delayed offer of screening was acceptable to most respondents (78%). CONCLUSION: Separating genomic screening from the stressful diagnostic period is supported by families who demonstrate good knowledge and recall. Our results suggest delaying genomic screening should be trialed more widely.

Humans↗

PubMed2005

Computers in anesthesia and intensive care: lack of evidence that the central unit serves as reservoir of pathogens.

OBJECTIVE: Computers are becoming increasingly visible in operating rooms (OR) and intensive care units (ICU) for use in bedside documentation. Recently, they have been suspected as possibly acting as reservoirs for microorganisms and vehicles for the transfer of pathogens to patients, causing nosocomial infections. The purpose of this study was to examine the microbiological (bacteriological and mycological) contamination of the central unit of computers used in an OR, a surgical and a pediatric ICU of a tertiary teaching hospital. METHODS: Sterile swab samples were taken from five sites in each of 13 computers stationed at the two ICUs and 12 computers at the OR. Sample sites within the chassis housing of the computer processing unit (CPU) included the CPU fan, ventilator, and metal casing. External sites were the ventilator and the bottom of the computer tower. Quantitative and qualitative microbiological analyses were performed according to commonly used methods. RESULTS: One hundred and ninety sites were cultured for bacteria and fungi. Analyses of swabs taken at five equivalent sites inside and outside the computer chassis did not find any significant-number of potentially pathogenic bacteria or fungi. This can probably be attributed to either the absence or the low number of pathogens detected on the surfaces. CONCLUSION: Microbial contamination in the CPU of OR and ICU computers is too low for designating them as a reservoir for microorganisms.

Anesthesiology↗

PubMed2006

Toll-like receptor recognition of Toxoplasma gondii.

Toxoplasma gondii potently stimulates IFN-gamma production by both the innate and adaptive immune system as part of its host adaptation. This response is known to be dependent on an Myeloid Differentiation factor 88 signaling pathway used by Toll-like receptors (TLRs), a family of proteins involved in the recognition of microbial molecular patterns. In the following review, we summarise the evidence for specific TLR function in host resistance to T. gondii focusing on the recent discovery in the parasite of a profilin-like ligand that potently stimulates TLR11 and regulates the production of IL-12, a cytokine necessary for the protective IFN-gamma response. In addition, we discuss the hypothesis that TLR11 may have evolved as a general pattern recognition receptor for apicomplexan protozoa and that as highly conserved proteins associated with actin-based motility, profilins are logical ligand targets for this form of pathogen detection. Finally, we review the evidence for involvement of other TLR and TLR ligands in host resistance to T. gondii and discuss how such receptors might synergise with TLR11 in the innate response to the parasite.

Animals↗

PubMed2006

Type I interferons in host defense.

Type I interferons (IFNs) are a family of cytokines specialized to coordinate immunity to viruses and other intracellular infections. In the past several years, many of the receptors and signaling pathways that link pathogen detection to induction of type I IFNs have been identified and characterized. An integrated picture has emerged in which type I IFNs have essential functions in several seemingly disparate processes: they restrict viral spread by engaging machinery that ultimately cripples and kills infected cells, yet they are also positively linked to the activation and expansion of lymphocytes that are important for control of intracellular infections. These advances highlight the context-specific actions of type I IFNs and clarify the multiple points at which they are integrated into both innate and adaptive immunity.

Animals↗

PubMed2004

Comparison of TaqMan and Epoch Dark Quenchers during real-time reverse transcription PCR.

Several biotechnology companies have recently introduced novel quencher fluors for use with dual-labeled fluorogenic hydrolysis probes. The Epoch Dark Quencher trade mark fluorochrome consists of a non-fluorescent moiety capable of absorption at higher wavelengths (400-650 nm). The aim of this study was to: (1) evaluate the feasibility of using Epoch Dark Quencher fluorochromes in real-time PCR pathogen detection assays that were previously optimized with TaqMan (TAMRA) quenching fluors, and (2) compare the sensitivity based on cycle threshold (CT) between probes containing either TaqMan or Epoch Dark Quencher fluors. Our data indicate Epoch Dark Quencher probes can be used in place of TaqMan probes and their performance was not better than traditional TaqMan (TAMRA) quenchers. Marginal differences observed between quenching fluorochromes may arise from concentration differences during probe synthesis.

Fluorescent Dyes↗

PubMed2005

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum.

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes (vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.

Animals↗

PubMed2006

Rapid and simple detection of food poisoning bacteria by bead assay with a microfluidic chip-based system.

A rapid bead assay for detecting pathogenic bacteria with a simple microfluidic chip-based system was developed. Five oligonucleotide probes corresponding to the 16S rRNA of the targeted bacteria were coupled covalently to fluorescent beads. Four species of bacteria (Escherichia coli, Salmonella enterica subsp. enterica serovar Enteritidis, Yersinia enterocolitica, and Bacillus cereus) were used as representative food-borne pathogenic bacteria. The RNAs extracted from pure cultures of these microorganisms were fluorescently labeled and hybridized to the oligonucleotide probes-immobilized fluorescent beads (Bead assay). The duplexes of RNAs and the probes-immobilized beads were analyzed with the commercially available microfluidic chip-based system. This bead assay provided results within 3 h following RNA extraction from bacterial cells.

Bacillus cereus↗

PubMed2005

Plants and animals: a different taste for microbes?

Plants and animals can recognize potential pathogens by detecting pathogen-associated molecular patterns (PAMPs). Significant advances over the past few years have begun to unveil the molecular basis of PAMP perception by pattern recognition receptors (PRRs). Although these discoveries highlight common recognition strategies among higher eukaryotes, they also show differences with respect to the nature of the receptors involved and the exact molecular patterns recognized. This suggests a convergent evolution of microbe sensing by the innate immune systems of these various organisms.

Animals↗

PubMed2006

Evolution of parasitic symbioses between plants and filamentous microorganisms.

Innate defense in wild plant populations is an invaluable resource for understanding how sustainable disease control can be achieved in crops through research that is rooted in molecular and evolutionary biology. Much progress has been made from molecular research into pathogen detection and defense induction. Bacterial pathology of the wild species Arabidopsis thaliana is at the forefront in revealing parallels with animal innate immunity against infectious diseases. In plants, unlike in animals, however, expansion in biodiversity has been mirrored by tremendous diversification in filamentous parasites. The fungal and oomycete pathology of Arabidopsis is exposing opportunities to investigate the molecular bases of compatibility, plant-driven speciation of parasites, and molecular epidemiology. Such research might reveal evidence that an arms race did occur in the evolution of plant-parasite symbioses.

Plant Diseases↗