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PubMed2026

Development and clinical validation of a CRISPR/Cas9-engineered reporter phage cocktail for rapid detection of Escherichia coli in urine.

Urinary tract infections are one of the most common infectious diseases, with Escherichia coli as the predominant pathogen. Traditional diagnostic methods fail to meet clinical demands for rapid and specific detection. Here, we developed an efficient urine E. coli detection strategy via a reporter phage cocktail. Four reporter phages (T2::Nluc, T4::Nluc, T5::Nluc, T6::Nluc) were constructed by the CRISPR/Cas9 system combined with homologous recombination. One-step growth curves, optimal multiplicity of infection, and lytic efficiency showed that the Nluc gene block insertion exerted heterogeneous effects on phages. Luminescence assays demonstrated that all five reporter phages (including previously preserved T7::Nluc) and the cocktail offered favorable limits of detection (≥103 CFU/mL), high specificity, and no urine matrix interference. However, single phages exhibited limited coverage among 177 clinical E. coli isolates. But the reporter phage cocktail remedies this limitation. In large-scale clinical validation, the cocktail achieved sensitivity 73.15% (63.76%-81.22%), specificity 100.00% (99.53%-100.00%), positive predictive value (PPV) 100.00% (95.44%-100.00%), and negative predictive value (NPV) 96.42% (95.18%-97.36%) (all 95% confidence interval [CI]), and excellent concordance with the gold-standard method (Kappa = 0.83, 95% CI: 0.77-0.89), greatly outperforming single reporter phages (~40.00% sensitivity). This method requires no sample pretreatment, is simple to operate, and completes detection within 4 h, significantly improving diagnostic efficiency. Accordingly, it provides a novel platform for pathogen detection and supports the clinical translation of reporter phage diagnostics.IMPORTANCEUrinary tract infections impose substantial economic and public health burdens. In this study, we successfully constructed Escherichia coli-specific reporter phages T2::Nluc, T4::Nluc, T5::Nluc, and T6::Nluc. Combined with the previously preserved T7::Nluc, these phages formed a reporter phage cocktail. Co-cultivation of this cocktail with clinical samples enabled rapid and specific detection of E. coli in clinical urine, with a significantly shortened detection time (4 h) and good concordance with the gold-standard detection method (Kappa = 0.83), effectively improving detection efficiency and accuracy. This novel pathogen detection platform, integrating specific recognition and signal amplification, not only provides a new technical approach for the rapid and accurate diagnosis of clinical urinary tract infections but also effectively promotes the coordinated improvement of infectious disease diagnosis and treatment in terms of timeliness-precision-cost.

Escherichia coli↗

PubMed1992

Characterization of a cryptic gene pair from Neisseria gonorrhoeae that is common to pathogenic Neisseria species.

A pair of genes, each of which produces in Escherichia coli a 20-kDa, periplasmically localized protein that cross-reacts with anti-rpoN monoclonal antibody, was isolated from Neisseria gonorrhoeae. Homologs of the two genes were detected in pathogenic Neisseria species but not in commensal species. These genes are designated cnp1 and cnp2 (cryptic neisserial protein).

Amino Acid Sequence↗

PubMed2006

Shigella as a foodborne pathogen and current methods for detection in food.

Shigella, the causative agent of shigellosis or "bacillary dysentery," has been increasingly involved in foodborne outbreaks. According to the Centers for Disease Control and Prevention's Emerging Infections Program, Foodborne Diseases Active Surveillance Network (FoodNet), Shigella was the third most reported foodborne bacterial pathogen in 2002. Foods are most commonly contaminated with Shigella by an infected food handler who practices poor personal hygiene. Shigella is acid resistant, salt tolerant, and can survive at infective levels in many types of foods such as fruits and vegetables, low pH foods, prepared foods, and foods held in modified atmosphere or vacuum packaging. Survival is often increased when food is held at refrigerated temperatures. Detection methods for Shigella include conventional culture methods, immunological methods, and molecular microbiological methods. Conventional culture of Shigella in foods is often problematic due to the lack of appropriate selective media. Immunological methods for Shigella have been researched, yet there is only one commercially available test kit. Molecular microbiological methods such as PCR, oligonucleotide microarrays, and rep-PCR have also been developed for the detection and identification of Shigella. This manuscript reviews the general characteristics, prevalence, growth and survival, and methods for detection of Shigella in food.

Dysentery, Bacillary↗

PubMed2000

Short communication: survival of Escherichia coli O157:H7 in dairy cattle drinking water.

Cattle drinking water from two dairy farms was used in a study to determine the survival characteristics of the bacterial pathogen Escherichia coli O157:H7 and wild-type E. coli. The E. coli O157:H7 inoculum consisted of a consortium of isolates obtained from dairy cattle. Fresh manure was used as the source for the wild-type E. coli. In the water source from farm 1 the pathogens were present at both 5 and 15 degrees C during the 16-d duration of the study. In the water source from farm 2, the pathogens were detected at 5 degrees C through d 8 and through d 4 at 15 degrees C. The fecal indicator, wild-type E. coli, was always present when the pathogens were present.

Animals↗

PubMedDate not supplied

Paramyxovirus type 3 (PMV-3) in California turkeys: serologic study of PMV-3 antibody with an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting paramyxovirus type 3 (PMV-3) antibody. This test had a higher sensitivity than the hemagglutination-inhibition test, and no cross-reactivity with various paramyxoviruses or avian pathogens was detected when the sera were tested diluted 1:200. The incidence of PMV-3 infection in California was studied by testing 2037 turkey sera from 174 meat and breeder flocks for the presence of PMV-3 antibody using the ELISA. The age at which the infection occurs was around 5 to 8 weeks for meat flocks and 10 to 12 weeks for breeder flocks. Infection with the PMV-3, as determined serologically, was more frequent than manifested cases of disease, and 95.2% of the flocks aged over 11 weeks had PMV-3 antibody. No typical manifestations of PMV-3 disease (respiratory disease plus drop in egg production) were observed, probably because of the early infection which occurred before laying age.

Animals↗

PubMed1996

[Prevalence of Chlamydia trachomatis among men consulting in urology. Comparative study between cell culture and sperm PCR].

OBJECTIVE: Genital Chlamydia trachomatis infection can be difficult to diagnose, depending on the precise site of infection. Given the lack of major clinical signs and symptoms in many cases and the risk of male and female infertility. Chlamydia trachomatis is a public health problem. It can be difficult to detect this pathogen in sperm by means of cell culture, because of seminal fluid toxicity for cell lines. New techniques such as the polymerase chain reaction (PCR) can be used to detect genomic DNA. STUDY DESIGN: We studies 81 patients by applying the Amplicor CT PCR test to sperm, in comparison with cell culture on sperm and urethral samples. RESULTS: The prevalence of Chlamydia trachomatis infection was not significantly different (3.7% vs 5%) in the urethral cell culture and PCR methods, respectively (p > 0.05). In contrast, PCR was significantly more sensitive than sperm cell culture (5% vs 1.2%; p < 0.03). Moreover, we have not detected of genital chlamydiose among the infertile men. CONCLUSION: These findings suggest that PCR detection of Chlamydia trachomatis can dispense with the need for urethral sampling and cell culture in selected male patients.

Adult↗

PubMed2006

Specific electrochemical phage sensing for Bacillus cereus and Mycobacterium smegmatis.

The rapid and reliable detection of pathogenic microorganisms is an important issue for the safety and security of our society. Here we describe the use of a sensitive, inexpensive, amperometric, phage-based biosensor for the detection of extremely low concentrations of Bacillus cereus and Mycobacterium smegmatis as models for Bacillus anthracis (the causative agent of anthrax) and for Mycobacterium tuberculosis (the causative agent of tuberculosis), respectively. The detection procedure developed here enabled the determination of bacteria at a low concentration of 10 viable cells/mL within 8 h. This experimental setup allows the simultaneous analysis of up to eight independent samples, using disposable screen-printed electrodes.

Bacillus cereus↗

PubMed1991

[A study on diarrhoea disease caused by Vibrionaceae along coast the east of Zhejiang Province].

A study of the etiology of 1028 cases of acute diarrhoea due to vibrionaceae was carried out between August 1988 to July 1990 along the coast of east Zhejiang Province. From 417 clinical cases (40.56%) were found. 590 strains (57.39%) of pathogenic vibrios Among them, the detection rates of vibrio, aeromonas and plesionmonas were 48.14%, 44.74%, 7.12% respectively. There were 8 Species of vibrio detected: V. parahemolyticus (63.73%), V. fluvialis (11.97%), V. alginolyticus (10.215), non-01 vibrio cholera (9.86%), V. furnissii (2.47%), V. mimicus (1.06%), V. metchnikovii (0.35%), V. vulnificus (0.35%). The detection rate of aeromonas species of A. sorbia, A. caviae, A. hydrophila and A. indefinite sp. were 30.68%, 27.27%, 22.73%, and 19.32% respectively. Plesionmonas shigellides was also found in this study. The age, sex, clinical features, detection rates of pathogenic vibrios of diarrhoea cases and sea food contact history were analyzed.

Adult↗

PubMed1991

The relationship of microbial pathogens to acute infectious diarrhoea of childhood.

Bacterial, viral and parasitic enteric pathogens were detected in 692 of 916 children below 36 months of age with acute diarrhoea and in 289 of 587 matched controls. The rates of identification of only four groups of pathogens, rotavirus, Shigellae, Salmonella typhimurium and enterotoxigenic E. coli, were significantly higher in the patients. The prevalence of a variety of other enteric pathogens was similar in controls of patients. Shigellosis had a characteristic clinical profile but none of the other agents could be suspected on clinical grounds. The high prevalence of pathogens in controls suggested that the population may be partially protected against a variety of enteric pathogens and that final common pathways leading to diarrhoea may be activated by changes in the microbial ecology of the gut lumen.

Acute Disease↗

PubMed1997

[Monitoring of inhibitors of the polymerase chain reaction for the detection of hepatitis C virus using the positive internal control].

In the direct detection of pathogens by polymerase chain reaction (PCR) from clinical samples, false negative results due to the presence of inhibitor are problematic. In order to monitor such an inhibitor, we evaluated the detection of the positive internal control in PCR assay for HCV-RNA in serum samples positive for anti-HCV antibodies, of which 41 samples were positive for HCV-RNA by competitive RT-PCR assay. The positive internal control was coamplified with HCV-RNA and hybridized to the specific probe on magnetic beads and then hybrids were detected by colorimetric measurement using automatic PCR machine (COBAS AMPLICOR). Detection of the positive internal control was negatively correlated with viral copy number in sera assayed by competitive RT-PCR. Five of 52 samples (9.6%) with high HCV-RNA copy number (10(7) or 10(8) copies/ml) showed negative results for the internal control. The negative results for the internal control turned out to be positive when the sera were diluted and re-assayed, suggesting competitively inhibitory effects of high viral copy number on amplification of the internal control. Addition of heparin in the serum sample showed an inhibitory effect with a dose dependent manner on the detection of both HCV-RNA and the internal control, with a more effect on the lower copy number of HCV. On the other hand, addition of hemoglobin in the sample with concentration of up to 450mg/dl had no inhibitory effect on the detection of either HCV-RNA or the internal control. Coamplification and detection of the positive internal control was demonstrated to be useful to estimate effects of inhibitors, which may be present in clinical samples, in the detection of HCV-RNA by PCR.

False Negative Reactions↗

PubMed1996

Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.

Animals↗

PubMed2025

Unraveling the complex genetic landscape of OTOF-related hearing loss: a deep dive into cryptic variants and haplotype phasing.

BACKGROUND: Pathogenic variants in OTOF are a major cause of auditory synaptopathy. However, challenges remain in interpreting OTOF variants, including difficulties in confirming haplotype phasing using traditional short-read sequencing (SRS) due to the large gene size, the potential incomplete penetrance of certain variants, and difficulties in assessing variants at non-canonical splice sites. This study aims to revisit the genetic landscape of OTOF variants in a Taiwanese non-syndromic auditory neuropathy spectrum disorder (ANSD) cohort using a combination of sequencing technologies, predictive tools, and experimental validations. METHODS: We performed SRS to analyze OTOF variants in 65 unrelated Taiwanese patients diagnosed with non-syndromic ANSD, complemented by long-read sequencing (LRS) for haplotype phasing. A prediction-to-validation pipeline was implemented to assess the pathogenicity of cryptic variants using SpliceAI software and minigene assays. RESULTS: Biallelic pathogenic OTOF variants were identified in 33 patients (50.8%), while monoallelic variants were found in five patients. Three novel variants, c.3864G > A (p.Ala1288 =), c.4501G > A (p.Ala1501Thr), and c.5813 + 2T > C, were detected. The pathogenicity of two non-canonical mis-splicing variants, c.3894 + 5G > C and c.3864G > A (p.Ala1288 =), was confirmed by minigene assays. LRS-based haplotype phasing revealed that the common missense variant c.5098G > C (p.Glu1700Gln) and the novel variant c.5975A > G (p.Lys1992Arg) are in cis and form a founder pathogenic allele in the Taiwanese population. CONCLUSIONS: Our study highlights the genetic heterogeneity of DFNB9 and emphasizes the importance of population-specific variant interpretation. The integration of advanced sequencing technologies, predictive algorithms, and functional validation assays will improve the accuracy of molecular diagnosis and inform personalized treatment strategies for individuals with DFNB9.

Humans↗

PubMed2001

[Recent progress in molecular diagnosis of deep-seated mycoses].

Diagnosing deep-seated mycoses continues to be a major challenge for the clinician. The non-culture-dependent laboratory assay with high sensitivity and specificity are needed for rapid diagnosis of deep-seated mycoses. Future clinical mycology laboratories will increasingly utilize DNA-based methods for the recognition of pathogenic fungi in patient specimens and for the identification of fungal isolates. Over the last ten years, increasing numbers of papers were published which document the molecular biological methods feasible to detect fungus-specific DNA sequence in clinical specimens. The polymerase chain reaction(PCR) and internal probes are central to these procedures. More recently, the non-isotopic in situ technique has been applied in the detection of pathogenic fungi. These methods have the potential to improve diagnostic accuracy and hasten the institution of specific antifungal therapy. This article will review some of the recent advances in molecular diagnosis of fungal infections.

DNA Probes↗

PubMed1989

Rotaviral diarrhoea and its treatment in a foal.

A severely dehydrated foal with a history of acute diarrhoea was presented to Massey University for treatment. Rotavirus was demonstrated in the faeces by electron microscopy. The failure to detect other pathogens suggested that rotavirus was the primary aetiological agent. Successful treatment of this case is described and the principles of treatment for diarrhoea, particularly fluid therapy, are reviewed. Antibodies to rotavirus were detected in the sera of nine mares and their foals on the stud that referred the case, indicating that rotaviral infection was probably endemic.

Journal Article↗

PubMed2001

Solid-phase capture of proteins, spores, and bacteria.

Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 microg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25 degrees C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.

Antibodies, Monoclonal↗

PubMed2004

Multi-faceted, multi-versatile microarray: simultaneous detection of many viruses and their expression profiles.

There are hundreds of viruses that infect different human organs and cause diseases. Some fatal emerging viral infections have become serious public health issues worldwide. Early diagnosis and subsequent treatment are therefore essential for fighting viral infections. Current diagnostic techniques frequently employ polymerase chain reaction (PCR)-based methods to quickly detect the pathogenic viruses and establish the etiology of the disease or illness. However, the fast PCR method suffers from many drawbacks such as a high false-positive rate and the ability to detect only one or a few gene targets at a time. Microarray technology solves the problems of the PCR limitations and can be effectively applied to all fields of molecular medicine. Recently, a report in Retrovirology described a multi-virus DNA array that contains more than 250 open reading frames from eight human viruses including human immunodeficiency virus type 1. This array can be used to detect multiple viral co-infections in cells and in vivo. Another benefit of this kind of multi-virus array is in studying promoter activity and viral gene expression and correlating such readouts with the progression of disease and reactivation of latent infections. Thus, the virus DNA-chip development reported in Retrovirology is an important advance in diagnostic application which could be a potent clinical tool for characterizing viral co-infections in AIDS as well as other patients.

Acquired Immunodeficiency Syndrome↗

PubMed1991

S-layer positive motile aeromonads isolated from channel catfish.

Motile aeromonads are ubiquitous aquatic bacteria that can cause motile aeromonad septicemia (MAS), a disease which affects channel catfish and can produce significant economic loss. Motile aeromonads isolated from commercially-raised channel catfish were screened for production of S-layer protein in order to evaluate its potential role in natural epizootics. The S-layer protein was produced by 14 of 24 (58%) isolates from epizootics evaluated in this study. Concomitant infections with other internal pathogens were detected in 10 of the 24 cases used in this study, and only one of those 10 isolates (10%) produced the S-layer protein. When Aeromonas sp. was the only internal pathogen diagnosed, 13 of 14 (93%) isolates produced the S-layer protein.

Aeromonas↗

PubMedDate not supplied

Tetracycline fibres used to control bacterial infection during guided tissue regeneration (GTR).

AIMS: The colonization of suspected periodontal pathogens during the healing of periodontal defects treated by guided tissue regeneration (GTR) with e-PTFE membranes and tetracycline fibers was investigated. METHODS: Fifteen patients, each with one pair of angular periodontal bone defects of comparable size and morphology were recruited for the study. In a matched-pair study design, the test defects were treated with e-PTFE membranes in combination with tetracycline fibers, while control defects were treated with e-PTFE membranes alone. Microbiological specimens were taken from control as well as from test sites preoperatively (T0), intraoperatively (T1), two weeks after surgery (T2) and from membranes at time of removal (T3). Cultural methods were used to identify the following species: Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn) and Actinobacillus actinomicetemcomitans (Aa). RESULTS: At baseline and T1, none of the suspected periodontal pathogens were detected at test or control sites. Levels of P. intermedia and F. nucleatum, as mean percentages of total culturable microflora, were detected at levels significantly higher in control sites than test sites at times T2 and T3. CONCLUSIONS: Findings from this study suggest that e-PTFE membranes are frequently colonized by periodontal pathogens, and that bacterial colonization of healing sites after GTR procedures can be kept under control with a controlled delivery system releasing tetracycline.

Aggregatibacter actinomycetemcomitans↗