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PubMed1994

Role of different methods for the detection of diarrhoeal pathogens in HIV-infected patients in Brazil.

To evaluate the ability of different diagnostic methods for the detection of AIDS-related diarrhoeal pathogens in developing countries, we studied 40 HIV-infected patients with diarrhoea. All patients were subjected to stool examinations for parasites, stool culture and peroral jejunal biopsy. Jejunal specimens were processed for histological examination with several stains and for transmission electron microscopy (TEM). Jejunal juice and mucosa were cultured. An aetiologic agent was found in twenty patients. Eleven stool specimens were positive for parasites and stool culture was positive in three patients. The enteropathogens detected by these two methods included every microorganism amenable to treatment. Histological examination revealed four agents not previously identified. TEM added to diagnosis in only two patients. All cultures of jejunal mucosa and jejunal juice were negative, even when stool culture was positive. We conclude that a minimal investigation consisting of stool examination for parasites and stool culture is a cost-effective strategy in the management of AIDS-related diarrhoea in developing countries.

AIDS-Related Opportunistic Infections↗
PubMed2003

Detection of bacterial pathogen DNA using an integrated complementary metal oxide semiconductor microchip system with capillary array electrophoresis.

In this paper, we show an integrated complementary metal oxide semiconductor (CMOS)-based microchip system with capillary array electrophoresis (CAE) for the detection of bacterial pathogen amplified by polymerase chain reaction (PCR). In order to demonstrate the efficacy of PCR reaction for the heat-labile toxin producing enterotoxigenic Escherichia coli (E. coli), which causes cholera-like diarrhea, 100 bp DNA ladders were injected along with the PCR product. Poly(vinylpyrrolidone) (PVP) was used as the separation medium and provided separation resolution which was adequate for the identification of PCR product. The miniaturized integrated CMOS microchip system with CAE has excellent advantages over conventional instrumental systems for analysis of bacterial pathogens such as compactness, low cost, high speed, and multiplex capability. Furthermore, the miniaturized integrated CMOS microchip system should be compatible with a variety of microfabricated devices that aim at more rapid and high-throughput analysis.

DNA, Bacterial↗
PubMed1986

Approaches to the detection of enteric pathogens, including Campylobacter, using nucleic acid hybridization.

A previous study of the detection of Campylobacter jejuni in fecal samples spotted directly onto nitrocellulose filters before hybridization revealed a relatively low sensitivity and some false-positive results. We have investigated two factors that interfere with the detection of Campylobacter jejuni in fecal samples: interfering substances that create false-positive background signals and nonspecificity of the probe. Heterologous deoxyribonucleic acid probes bound nonspecifically to partially extracted fecal samples, indicating that the major basis for false-positive background was not due to homologous sequences. The problem of false-positive results could be reduced, but not eliminated, by extensive deoxyribonucleic acid extraction procedures applied to the clinical sample. The relative concentration of protein in each sample may be an important contributor to nonspecific binding. Other measures, such as sonication, glass bead fragmentation, and column separation, were not helpful. Development of a species-specific deoxyribonucleic acid probe derived from sequences encoding 16S ribonucleic acid is underway.

Campylobacter Infections↗
PubMed2005

Detection and recovery rates achieved using direct plate and enrichment/immunomagnetic separation methods for Escherichia coli O157:H7 in minced beef and on bovine hide.

AIMS: To assess the detection and recovery rates achieved with commonly used cultural methods for the enumeration and recovery of Escherichia coli O157:H7 from minced beef and bovine hide. METHODS AND RESULTS: Minced beef and bovine hide were inoculated with varying concentrations (log(10) 1.58-2.58 CFU g(-1) and log(10) 2.42-4.49 CFU 100 cm(2) respectively) of E. coli O157:H7 and recovered using a direct plate method or an enrichment/immunomagnetic separation (IMS) method and then plated onto SMAC or SMAC-CT in both cases. The direct plate method detected the pathogen consistently from minced beef samples with an average recovery of 69.2-91.2%. From faecal material on the bovine hide the recovery of the pathogen ranged from 1.80 to 64.5% with fresh faeces depending on the inocula while from dried faeces on hide the results ranged from no recovery at all to 25.1%. Enrichment/IMS recovered E. coli O157:H7 at all inocula levels tested in minced beef while the pathogen was only detected consistently at an average inocula level of log(10) 2.73 CFU 100 cm(2) from fresh faeces and log(10) 4.49 CFU 100 cm(2) from dried faeces on bovine hide. CONCLUSIONS: The direct count enumeration method for E. coli O157:H7 underestimated the numbers of pathogens present. The enrichment/IMS procedure consistently detected the pathogen from minced beef but did not always detect E. coli O157:H7 from faeces on bovine hide. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall this study highlights that any microbial data, used in either predictive microbiology or risk assessment, must take account of the sensitivity and associated performance of the methods employed, in order to make an accurate reflection of the true microbiology of the examined sample.

Animals↗
PubMed2006

Comparison of different protocols for DNA preparation and PCR for the detection of fungal pathogens in vitro.

Although a large number of different PCR protocols for the detection of fungal DNA from clinical samples have been described, a generally recognised standardisation has not yet been developed. In a first step, we compared six different methods to isolate DNA under in vitro conditions from Aspergillus fumigatus, Candida albicans and Saccharomyces cerevisiae with respect to efficiency and expenditure of time. To this end, methods were tested that are based on both mechanical and enzymatic/thermic lysis. Thereby, enzymatic/thermic lysis were shown to be superior to mechanical lysis, although these methods of DNA isolation were more time consuming. The subsequent comparison of three different PCR protocols showed real-time PCR to be the most sensitive method.

DNA, Fungal↗
PubMed2026

Comparative evaluation of probe-capture and conventional metagenomic sequencing across multiple clinical sample types, with analysis of paired bronchoalveolar lavage fluid and blood samples.

Conventional metagenomic next-generation sequencing (mNGS) suffers from host nucleic acid interference and poor performance in low-biomass samples. Probe-capture metagenomic sequencing (PC-mNGS), which enriches microbial targets via hybridization probes, shows superior sensitivity but lacks systematic multi-sample evaluations. This study compared PC-mNGS and mNGS across diverse clinical specimens (bronchoalveolar lavage fluid [BALF], blood, cerebrospinal fluid [CSF]) and assessed the clinical utility of pathogen co-detection in paired BALF-blood samples from sepsis patients. A total of 282 samples (81 BALF, 141 blood, 25 CSF, 35 others) sequenced by both PC-mNGS and mNGS were analyzed. Additionally, 621 paired BALF-blood samples from sepsis patients with pulmonary infections were evaluated. PC-mNGS achieved higher pathogen detection rates (66.67% vs 57.10%, P = 0.000198) than mNGS, particularly in blood (66.67% vs 47.52%, P = 2.5 × 10⁻⁵). PC-mNGS detected more bacteria (19 species exclusive) and fungi (11 species exclusive) than mNGS. Viruses showed comparable detection. BALF and CSF exhibited high overall agreement (OPA: 96.30% and 88%, respectively), while blood had lower concordance (NPA: 54.05%, OPA: 70.92%). A total of 60.55% of BALF-positive samples (PC-mNGS) had co-detected pathogens in blood. Gram-negative bacteria (e.g., Klebsiella pneumoniae) and fungi (e.g., Candida albicans) showed higher blood co-detection rates than viruses. In this study, PC-mNGS detected more pathogens and showed a higher positivity rate than mNGS in blood samples. BALF sequencing data, particularly bacterial reads per million (RPM), may predict bloodstream co-detection, aiding in sepsis management. However, clinical validation and integration with traditional diagnostics are needed to confirm utility. This study highlights PC-mNGS as a promising tool for complex infections but underscores the need for rigorous multi-context validation.IMPORTANCEAccurate and rapid identification of pathogens is critical for effective treatment of severe infectious diseases, such as sepsis. This study demonstrates that probe-capture metagenomic sequencing (PC-mNGS) detected more pathogens in blood samples compared to conventional metagenomic sequencing, especially for bacterial and fungal infections. By analyzing paired lung and blood samples, we show that high pathogen levels in lung fluid may predict bloodstream infection, offering a potential early warning for clinicians. These findings support the use of PC-mNGS as a more sensitive diagnostic tool, which could lead to faster, more targeted therapies and better outcomes for patients with complex infections.

Humans↗
PubMedDate not supplied

[Development of diagnostic kit "Ptakh-Grip PLR" for detection of highly pathogenic strain of avian influenza virus H5N1 by polymerase chain reaction].

A diagnostic kit for detection of avian influenza virus by real-time polymerase chain reaction was developed. This kit allows to identify the influenza viruses type A and highly pathogenic strain of avian influenza virus H5N1. The diagnostic kit is universal and adopted for ABI PRISM SDS (Applied Biosystems), RotorGene (Corbett Research) and iQCycler (BioRad) PCR machines.

Amino Acid Sequence↗
PubMed2006

Antibody-based surface plasmon resonance detection of intact viral pathogen.

Surface plasmon resonance (SPR) technique was used to directly detect an intact form of insect pathogen: the baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). An SPR sensor chip with three bio-functional layers was used to detect the intact AcMNPV: amine-reactive crosslinker with a disulfide bond that chemisorbs to gold film, Protein A, and a mouse IgG monoclonal antibody raised against a surface protein of the target viral pathogen. A two-channel (reference & test) micro-fluidic SPR system is used for reliable measurement. Bio-specific response to the AcMNPV is compared with the response for tobacco mosaic virus (TMV) as control. Successive exposure of the sensor chip to both viruses verifies a specific response to AcMNPV. This serves as a prerequisite to the development of a new type of viral pathogen detection sensors.

Antibodies, Viral↗
PubMed2005

DNA microarray-based detection of nosocomial pathogenic Pseudomonas aeruginosa and Acinetobacter baumannii.

Infection by nosocomial pathogenic bacteria is increasingly becoming a major threat to the patients in the hospital. We have developed a diagnostic DNA microarray for the detection of two important nosocomial pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii. The diagnostic DNA microarray contains the species-specific probes of 15mer oligonucleotides designed based on the sequences of 23S ribosomal DNA. The performance of DNA microarray in diagnosing P. aeruginosa and A. baumannii was evaluated using reference bacteria as well as clinical specimens such as blood, stool, pus, sputum, urine and cerebrospinal fluid. Using this DNA microarray, A. baumannii could be successfully detected in 11 out of 13 clinical specimens, thus giving the sensitivity of 84.6% with the specificity of 100% and the positive predictive value of 100%. P. aeruginosa could also be detected in 25 out of 26 clinical specimens, showing the sensitivity of 96.2%, the specificity of 100%, and the positive predictive value of 100%. These results suggest that two nosocomial pathogens, P. aeruginosa and A. baumannii, can be efficiently diagnosed by using the DNA microarray developed in this study.

Acinetobacter baumannii↗
PubMed2006

The prospects of advanced molecular methods in the detection of periodontal pathogens: hybridization in situ.

The paper summarizes the present state in the diagnostics of periodontal pathogens. Both the main advantages and drawbacks of the classic cultivation methods and those of the new DNA techniques are discussed. From the emerging methods of molecular diagnostics, the method of in situ hybridization is presented in more details. Its principle, various modifications of performance and possibilities of use are explained, including examples of its application in the detection of periodontal pathogens.

Bacteria↗
PubMed1996

Acanthamoeba: ecology, pathogenicity and laboratory detection.

Acanthamoeba spp. are ubiquitous free-living protozoa found in a wide range of environmental niches. They are resistant to disinfectants, temperature variation and desiccation and are responsible for two recognised diseases in humans, granulomatous amoebic encephalitis and keratitis. Both infections are rare, although the latter is currently receiving more attention following the association between Acanthamoeba and the wearing of contact lenses. Laboratory diagnosis is unusual but not beyond the bounds of most routine clinical microbiology departments. In this review the various aspects surrounding the ecology, pathogenicity and laboratory detection of Acanthamoeba spp. are considered.

Acanthamoeba↗
PubMed1996

Volume of blood required to detect common neonatal pathogens.

OBJECTIVE: To determine the minimum volume of blood and the absolute number of organisms required for detection of bacteremia and fungemia by an automated colorimetric blood culture system (BacT/Alert, Organon Teknika). DESIGN: Common neonatal pathogens, Escherichia coli, Streptococcus agalactiae (group B streptococcus (GBS): one American Type Culture Collection (ATCC) strain and one clinical isolate), Staphylococcus epidermidis, and Candida albicans, were seeded into blood to produce bacteremia or fungemia with low colony counts (1 to 3 colony-forming units (CFU) per milliliter) and ultra-low colony counts (<1 CFU/ml). For each organism, 96 culture bottles were inoculated with either 0.25, 0.5, 1.0, or 4.0 ml of the two seeded blood concentrations. Blood culture bottles were incubated in the BacT/Alert device for 5 days, and time to positivity was noted when applicable. All bottles were subcultured on plated media. DATA ANALYSIS: The Poisson statistic was used to calculate the probability of finding at least one viable CFU per inoculated culture bottle. The fraction of culture bottles with positive findings per group was divided by the probability of one or more organisms present to give the positivity index. RESULTS: Plated subculture identified no growth of organisms not detected by the colorimetric detection system. The false-positive rate for the automated device was less than 1%. The positivity index for the GBS clinical isolate was 1.13, for the GBS ATCC isolate 0.96, for S. epidermidis 0.94, for C. albicans 0.97, and for E. coli 0.95. There was a statistically significant difference with time to positivity and inocula volume (p <0.01), but the difference was not clinically important. CONCLUSIONS: If one or two viable colony-forming units are in the blood inoculated into culture media, the BacT/Alert system will detect growth rapidly. Because there appears to be a sizable subset of neonates who are at risk of sepsis with a colony count less than 4 CFU/ml, then a 0.5 ml inoculum of blood into the culture media is inadequate for sensitive and timely detection of bacteremia. One to two milliliters of blood should increase microorganism recovery in the face of low-colony-count sepsis.

Bacteremia↗
PubMed1997

Rapid molecular detection of microbial pathogens: breakthroughs and challenges.

Microbiological contamination of foods and drinking water is a global problem, and a significant amount of expense is being incurred as a result of such contamination. The microorganisms associated with almost half of all disease outbreaks still go unidentified, primarily as a result of inadequate monitoring and surveillance. Though significant improvements have been made in refining molecular methods for detecting infectious agents, a majority of these methods are being employed only on clinical samples where pathogen densities are much higher than those found in environmental and food samples. Comparative evaluations of the various protocols in terms of cost, sensitivity, specificity, speed, and reproducibility need to be undertaken so that the true applicability of these methods can determined. In the future, molecular methods, especially gene amplifications and in situ hybridizations, will find increasing applications in the differentiation of viable and non-viable organisms, in predicting antimicrobial resistance, and in the identification and characterization of unculturable microorganisms. Though molecular detection methods will not totally replace conventional methods, they will significantly enhance our ability to detect microbial pathogens rapidly.

Animals↗
PubMed1990

Detection of surgical pathogens by in vitro DNA amplification. Part I. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene.

The management of candidemia and disseminated candidiasis depends on rapid, unambiguous identification of Candida. Such identification is retarded by the slow growth of Candida from clinical specimens. Administration of effective but potentially toxic antifungal therapy is often withheld pending identification. To circumvent this slow growth and thus to expedite diagnosis and therapy, the polymerase chain reaction (PCR) was used to amplify a segment of fungal DNA coding for the cytochrome P450L1A1 (lanosterol-14 alpha-demethylase) in vitro. The technique provides unambiguous evidence of C. albicans in as few as 6 hours with a detection threshold of 10 organisms in a 100 mu specimen. Clinical specimens of urine (n = 4), sputum (n = 6), wound fluid (n = 1, and blood (n = 2) were collected from patients, and C. albicans was conventionally documented at these sites; in each case, PCR was confirmed. Of 17 additional specimens that were culture negative, PCR suggested the presence of yeast in two of the specimens. PCR-based detection of surgical pathogens may have broad application in rapid screening for the presence of organisms either indigenous to a particular surgical intensive care unit or peculiar to selected patient populations.

Candida albicans↗
PubMed1997

Comparison of the BacT/Alert FAN aerobic and the Difco ESP 80A aerobic bottles for pediatric blood cultures.

We compared the BacT/Alert system using the aerobic FAN bottle with the ESP system using the 80A aerobic bottle for the detection of pediatric bloodstream pathogens at a children's hospital. From 6,636 blood culture sets complying with the inclusion criteria, 308 pathogens were detected, including 177 that were detected by both systems, 69 that were detected by BacT/Alert FAN only, and 62 that were detected by ESP 80A only (P = 0.6; not significant). BacT/Alert FAN detected more isolates of Staphylococcus aureus (47 versus 34; P = 0.02), while ESP 80A detected more episodes of streptococcal and enterococcal infection. BacT/Alert FAN detected more pathogens from patients receiving antibiotic therapy (107 versus 93; P = 0.04). Of 248 separate episodes of bacteremia or fungemia, 146 were detected by both systems, 56 were detected by ESP 80A only, and 46 were detected by BacT/Alert FAN only (P = 0.37; not significant). The median times to detection were 13.6 h for ESP 80A and 15.7 h for BacT/Alert FAN (P < 0.001). Both systems were considered easy to operate and were free from significant mechanical difficulties. False-positive or false-negative signals were rare or nonexistent with both systems. We conclude that both systems rapidly detect a broad range of pediatric bloodstream pathogens. BacT/Alert FAN provides better detection of Staphylococcus aureus, especially from patients receiving antibiotics. ESP 80A provides better detection of streptococci and enterococci.

Bacterial Typing Techniques↗
PubMed2006

Influence of sampling technique on detection of potential pathogens in the nasopharynx.

OBJECTIVES: To determine the optimal approach for nasopharyngeal culture and to establish which approach children tolerate best. DESIGN: Cross-sectional study. SETTING: A pediatric otolaryngology department of a Dutch tertiary care hospital. PATIENTS: A cohort of 42 children with chronic suppurative otitis media. INTERVENTION: Paired nasopharyngeal samples were collected transorally and transnasally and cultured for potential aerobic pathogens. MAIN OUTCOME MEASURES: The isolation rate of both samples and the amount of discomfort measured by the visual analog scale. RESULTS: Forty-six (87%) of 53 samples obtained transnasally were culture positive vs 40 (75%) of 53 samples obtained transorally (P = .20). Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus were found more frequently with the transnasal than with the transoral approach: 34% vs 13% (P = .003), 62% vs 51% (P = .20), 30% vs 19% (P = .15), and 21% vs 11% (P = .18), respectively. Mean (SD) visual analog scale scores were 5.3 (1.0) and 3.4 (1.7) (P<.001) for the transnasal and transoral approaches, respectively. CONCLUSIONS: Although the transoral approach is better tolerated in children, the isolation rate of the transnasal approach is higher, especially for S. pneumoniae. The transnasal sampling technique should therefore be the preferred approach for detection of potential pathogens in the nasopharynx in children.

Bacteriological Techniques↗
PubMed1994

Rapid detection of food-borne pathogenic bacteria.

Recent advancements in biotechnology are rapidly altering the diagnostic procedures used in microbiologic analysis of foods. Biochemical identification tests have been miniaturized and automated, making them faster and more economical. Pathogenic bacteria that were previously isolated and identified after labor- and time-intensive enrichment and plating procedures can now be detected by measuring specific physicochemical changes resulting from their growth or metabolic activity. Nucleic acid and antibody-based assays are now used to rapidly and reliably detect pathogenic bacteria in foods. Nevertheless, foods offer unique challenges to the application of these techniques because of their complexity and variety, their interference with the rapid detection methods, and the need to detect pathogenic bacteria when they are present in foods at very low levels. Methods to sequester target pathogenic bacteria from interfering food components and to concentrate them in small volumes are needed to enable the efficient application of rapid detection and identification methods.

Bacteria↗