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PubMedDate not supplied

Quantification of human astroviruses in sewage using real-time RT-PCR.

Human astroviruses constitute a significant cause of acute diarrhea in children. Viral transmission occurs via the fecal-oral route, predominantly person-to-person, but the consumption of fecally contaminated water and shellfish has also been implicated. Viral pathogen detection in water, especially, in wastewater, is difficult and PCR is widely used to detect these viruses. Despite the recent development of real-time quantitative PCR, quantification of astroviruses in sewage had not been available up to now. We have developed a method to quantify astroviruses in sewage. For this purpose, we designed a set of primers and a fluorogenic probe located at the 3' -end of the genome of human astroviruses. The amplified region was cloned and the plasmid was transcribed to generate calibration standards for quantification. After validation of the standards, the method was evaluated in artificially contaminated samples. To validate the method on naturally contaminated samples, raw and treated wastewater samples were collected monthly for one year in a sewage treatment plant. Astrovirus genomes were detected in all samples collected at the entrance to the sewage treatment plant, with a mean value of 4.1 x 10(6) astrovirus genomes for 100 ml. Effluents were less strongly contaminated, with a mean value of 1.01 x 10(4) astrovirus genomes. The high prevalence of astroviruses in sewage treatment plant effluents indicates that these plants are not efficiently eliminating the virus. This is a major public health concern and new techniques of depuration are needed. Our method could be effectively used in evaluating new treatment processes to reduce the viral load in the effluent of treatment plants.

Environmental Monitoring↗

PubMed2026

Exploratory proteomic and metabolomic profiling of pleural effusions identifies histone H4 and alanine as promising complementary markers for pleural tuberculosis.

The diagnosis of pleural tuberculosis (Pl-TB) remains challenging. Histopathological analysis and pathogen detection in pleural biopsies are informative but limited. We investigated differentially expressed proteins and metabolites in pleural effusions from patients with Pl-TB, malignancies, and other pathologies. A proteomic analysis of pooled pleural effusions identified 45 proteins exclusively detected or upregulated in Pl-TB samples, many linked to infectious processes. Conversely, 18 proteins were uniquely found or upregulated in malignant pleural effusions, mainly associated with detoxification and hemostasis. To validate these findings, we employed targeted proteomics in individual samples. Eight proteins were validated: S100-A9, histone H4, insulin-like growth factor-binding protein 2, fibrinogen beta chain, ficolin-3, immunoglobulin heavy constant alpha 1, sulfhydryl oxidase 1, and histidine-rich glycoprotein. Additionally, NMR-based metabolomics identified 13 metabolites with differential abundance between Pl-TB and non-TB samples. Notably, N-acetyl-glycoprotein and the branched-chain amino acids, alanine and lysine differed between groups. Proteomic and metabolomic analyses revealed distinct molecular profiles between Pl-TB and non-TB patients, despite intra-group variability. To address this, we applied classification models. Histone H4 and alanine consistently emerged as discriminative features. Overall, this study provides novel insights into the molecular landscape of Pl-TB. The combined quantification of proteins and metabolites may improve differential diagnosis, although should be further validated in larger, independent cohorts before clinical application.

Humans↗

PubMed2006

The geographic distribution of the putative agent of epizootic bovine abortion in the tick vector, Ornithodoros coriaceus.

Epizootic bovine abortion (EBA), also known as "foothill abortion", is a vector borne disease of beef cattle that graze in the mountainous regions of California, southern Oregon and western Nevada transmitted by the argasid tick Ornithodoros coriaceus. Recently, the putative agent of EBA was identified as a novel Deltaproteobacter in the order Myxococcales. In this study, a TaqMan real-time PCR (TM-PCR) protocol specific to the putative EBA agent was developed. The new real-time TM-PCR assay functioned sensitively and specifically to detect pathogen DNA in field-collected O. coriaceus ticks. The assay had an analytical sensitivity of a single plasmid copy and, when evaluated with a collection of tick-borne pathogens, yielded a positive PCR-result only for the agent of EBA. Use of the TM-PCR represents an effective tool for rapid and highly sensitive assessment of environmental risk and spatial and statistical analysis to highlight areas where there may be increased risk for EBA in susceptible cattle.

Abortion, Veterinary↗

PubMed1999

Is Streptococcus pneumoniae the leading cause of pneumonia of unknown etiology? A microbiologic study of lung aspirates in consecutive patients with community-acquired pneumonia.

PURPOSE: Although a wide variety of recognized pathogens can cause community-acquired pneumonia, in many patients the etiology remains unknown after routine diagnostic workup. The aim of this study was to identify the causal agent in these patients by obtaining lung aspirates with transthoracic needle aspiration. SUBJECTS AND METHODS: During a 15-month period, all consecutive patients with community-acquired pneumonia who were eligible for transthoracic needle aspiration were enrolled in the study. In addition to conventional microbial methods (culture of blood and sputum, serologic studies), we performed cultures and genetic and antigen tests for common respiratory pathogens in lung aspirates. RESULTS: The study group consisted of 109 patients. Conventional microbial studies identified an etiology in 54 patients (50%), including Mycoplasma pneumoniae in 19 patients, Chlamydia pneumoniae in 9 patients, and Streptococcus pneumoniae in 9 patients. Among the remaining 55 patients, study of the lung aspiration provided evidence of the causal agent in 36 (65%). In 4 additional patients with a single microbial diagnosis by conventional methods, the lung sample provided evidence of an additional microorganism. The new pathogens detected by lung aspiration were S. pneumoniae in 18 patients, Haemophilus influenzae in 6 patients, Pneumocystis carinii in 4 patients, and C. pneumoniae in 3 patients; other organisms were identified in 4 patients. CONCLUSIONS: In our study, S. pneumoniae was the leading cause of community-acquired pneumonia, accounting for 25% of all cases, including about one-third of the cases the cause of which could not be ascertained with routine diagnostic methods.

Adult↗

PubMed1988

Rapid intravenous rehydration by means of a single polyelectrolyte solution with or without dextrose.

We compared the efficacy and safety of a single polyelectrolyte solution, Dhaka solution (DS), containing 133 mmol/L sodium, 13 mmol/L potassium, 98 mmol/L chloride, and 48 mmol/L acetate with and without 139 mmol/L (25 gm/L) dextrose in the rapid (4 hours) rehydration of 67 patients with diarrhea and moderate or severe dehydration requiring parenteral fluid therapy. Of the 67 patient, 31 were randomly assigned to receive the dextrose-containing solution (DS + D) and 36 DS without dextrose. On admission to the hospital, the two groups of patients were similar with respect to enteric pathogens detected, proportion with hyponatremia, magnitude of dehydration as assessed by clinical criteria, serum protein or creatinine concentration, and plasma glucose levels. At the end of the 4-hour infusion, both groups of patients had similar decreases in serum creatinine and protein levels and similar volume of urine output, but patients receiving DS + D had a significantly higher plasma glucose level than patients receiving DS (7.8 mmol/L (140 mg/dl) vs 5.39 mmol/L (97 mg/dl), P less than 0.01). One patient in the DS group had hypoglycemia (plasma glucose 2.0 mmol/L (36 mg/dl) at 4 hours. No other complications were noted. Serum protein values 24 hours after admission were little changed from 4-hour values, suggesting that rehydration was complete at the end of 4 hours. We conclude that, in our patients, rehydration can be carried out safely and rapidly with the use of a single solution and that adding 139 mmol/L (25 gm/L) of dextrose to the solution can prevent hypoglycemia without producing an osmotic diuresis.

Adolescent↗

PubMed1996

Azithromycin therapy for Cryptosporidium parvum infection in four children infected with human immunodeficiency virus.

Cryptosporidium parvum intestinal infection in immunodeficient patients can cause severe intestinal fluid losses with severe dehydration or chronic diarrhea with malnutrition. Therapies tried in human beings and animals include paromomycin, clarithromycin, azithromycin, octreotide, hyperimmune bovine colostrum, and bovine transfer factor. No specific therapy has been found to be consistently beneficial to children. We report azithromycin treatment of four children with acquired immunodeficiency syndrome who had severe diarrheal illnesses in which Cryptosporidium parvum was the sole pathogen detected. Three of these children had a marked decrease in stool volume and frequency within 36 hours of initiating therapy and resolution of diarrhea within 5 days; Cryptosporidium organisms became undetectable on examination of stool or colonic biopsy or by both after therapy was discontinued. A fourth patient required prolonged therapy with azithromycin to achieve clearance. Azithromycin therapy should be considered for immunocompromised patients with intestinal Cryptosporidium infection.

AIDS-Related Opportunistic Infections↗

PubMed2000

Invasive fungal sinusitis in the acquired immunodeficiency syndrome.

Invasive fungal sinusitis can present as either an indolent or fulminant process that primarily affects immunocompromised individuals. In this article, the clinical characteristics of four cases of invasive fungal sinusitis in patients with AIDS are analyzed and 22 additional previously reported cases in the literature are reviewed. In addition to HIV infection, other variables common to these cases include facial pain or headache out of proportion to clinical or radiographic findings, CD4 lymphocyte count less than 50 cells/mm(3), absolute neutrophil count less than 1,000 cells/mm(3), subtle radiographic evidence suggesting invasion and an indolent clinical course of the invasive infection. The most common pathogen detected was Aspergillus fumigatus. Maintaining a high index of suspicion, critically assessing these clinical findings, and prudently reviewing CT scans may facilitate early diagnosis and prompt intervention in these patients.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Plant disease resistance genes: recent insights and potential applications.

Plant disease resistance genes (R genes) encode proteins that detect pathogens. R genes have been used in resistance breeding programs for decades, with varying degrees of success. Recent molecular research on R proteins and downstream signal transduction networks has provided exciting insights, which will enhance the use of R genes for disease control. Definition of conserved structural motifs in R proteins has facilitated the cloning of useful R genes, including several that are functional in multiple crop species and/or provide resistance to a relatively wide range of pathogens. Numerous signal transduction components in the defense network have been defined, and several are being exploited as switches by which resistance can be activated against diverse pathogens.

Gene Expression Regulation, Plant↗

PubMed2000

Stressed salmonella are exposed to reactive oxygen species from two independent sources during recovery in conventional culture media.

Previously, Stephens et al. [J. Appl. Microbiol. 83 (1997) 445-455] developed a sensitive technique for studying the resuscitation of low levels of stressed Salmonella. Using this technique the influence on recovery performance of the peptone component of buffered peptone water was investigated. Within 12 different peptone types as much as 3.5 log10 cells/ml difference was observed between the best and worst performing formulations. Poor recovery performance was linked to reactive oxygen species (ROS) generation through auto-oxidation of reducing sugars and photo-sensitisation of sensitive components such as riboflavin. Supplementary recovery agents were explored with only Oxyrase, which has both enzymes to degrade ROS and the ability to rapidly turn a medium anaerobic, having any significant effect. It improved the speed of recovery and increased, by up to 100-fold, the number of stressed cells recovered. Stressed cells were further studied by flow cytometry with cell sorting, based on the staining pattern from a novel fluorochrome combination, into good and poor recovery media. It was identified that within a stressed population the removal of all oxygen protected actively respiring cells the most by forcing them to generate energy from anaerobic metabolism thus avoiding any risk from accidental endogenous ROS generation. The recognition of two independent sources of oxidative stress in the routine use of conventional culture media is discussed in relation to pathogen detection and other areas of food microbiology.

Carbohydrates↗

PubMed2000

Recombinant antibodies: a new reagent for biological agent detection.

Antibodies are critical reagents used in several biodetection platforms for the identification of biological agents. Recent advances in phage display technology allow isolation of high affinity recombinant antibody fragments (Fabs) that may bind unique epitopes of biological threat agents. The versatility of the selection process lends itself to efficient screening methodologies and can increase the number of antigen binding clones that can be isolated. Pilot scale biomanufacturing can then be used for the economical production of these immunoglobulin reagents in bacterial fermentation systems, and expression vectors with hexahistidine tags can be used to simplify downstream purification. One such Fab reagent directed against botulinum neurotoxin A/B has been shown to be sensitive in a variety of assay formats including surface plasmon resonance (SPR), flow cytometry, enzyme linked immunosorbent assay (ELISA), and hand-held immunochromatographic assay. Recombinant antibodies can provide another source of high quality detection reagents in our arsenal to identify or detect pathogens in environmental samples.

Animals↗

PubMed2004

Clinical usefulness of bronchoalveolar lavage in heart transplant recipients with suspected lower respiratory tract infection.

BACKGROUND: Bronchoscopy with bronchoalveolar lavage (BAL) is the recommended initial invasive diagnostic procedure when lower respiratory tract infection is suspected in solid-organ transplant recipients. In this study, we evaluated the clinical impact and safety of bronchoscopy with BAL in heart transplant recipients. METHODS: We reviewed all 44 consecutive diagnostic bronchoscopies with BAL that were performed in 35 heart transplant recipients at Helsinki University Central Hospital between May 1988 and December 2001. RESULTS: Bronchoscopy findings established specific microbiologic diagnoses in 18 of 44 (41%) cases, and 14 of 44 (32%) bronchoscopic findings led to changes in therapy. The diagnostic yield of bronchoscopy from 1 to 6 months after transplantation was 73%, significantly better (p = 0.002) than diagnostic yield during the first month (18%) and after 6 months (28%). Pneumocystis carinii and cytomegalovirus were the most frequently detected pathogens in the BAL fluid. Cytomegalovirus pneumonia carried a high mortality rate (44%), whereas all patients with P carinii pneumonia recovered. Fourteen episodes were diagnosed as bacterial pneumonia, but because of empiric antibiotic therapy that was started widely before bronchoscopy, a microbiologic diagnosis was established in only 1 case. However, all patients with community-acquired pneumonia responded to empiric therapy. Four cases of major complications occurred after bronchoscopy, all cardiovascular but none fatal. CONCLUSIONS: Bronchoscopy with BAL is a useful diagnostic tool in heart transplant recipients, especially between 1 and 6 months after transplantation.

Adolescent↗

PubMed1998

Global dinucleotide signatures and analysis of genomic heterogeneity.

Early biochemical experiments measuring nearest neighbor frequencies established that the set of dinucleotide relative abundance values (dinucleotide biases) is a remarkably stable property of the DNA of an organism. Analyses of currently available genomic sequence data have extended these earlier results, showing that the dinucleotide biases evaluated for successive 50 kb segments of a genome are significantly more similar to each other than to those of sequences from more distant organisms. From this perspective, the set of dinucleotide biases constitutes a 'genomic signature' that can discriminate sequences from different organisms. The dinucleotide biases appear to reflect species-specific properties of DNA stacking energies, modification, replication, and repair mechanisms. The genomic signature is useful for detecting pathogenicity islands in bacterial genomes.

Bacteria↗

PubMed2004

PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings.

Molecular diagnostics are revolutionising the clinical practice of infectious disease. Their effects will be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. PCR is the most well-developed molecular technique up to now, and has a wide range of already fulfilled, and potential, clinical applications, including specific or broad-spectrum pathogen detection, evaluation of emerging novel infections, surveillance, early detection of biothreat agents, and antimicrobial resistance profiling. PCR-based methods may also be cost effective relative to traditional testing procedures. Further advancement of technology is needed to improve automation, optimise detection sensitivity and specificity, and expand the capacity to detect multiple targets simultaneously (multiplexing). This review provides an up-to-date look at the general principles, diagnostic value, and limitations of the most current PCR-based platforms as they evolve from bench to bedside.

Communicable Diseases↗

PubMed1996

Microbiology of HIV associated bacteraemia and diarrhoea in adults from Nairobi, Kenya.

We undertook a retrospective descriptive comparison of the spectrum of pathogens responsible for bacteraemia and diarrhoea in HIV antibody positive and negative patients over 4 years (1988-92), in Nairobi, Kenya. The study population was recruited from primary to tertiary centres of clinical care and consisted of 2858 adults (15 years or older). There were 415 significant blood culture isolates, 192 from 1785 HIV negative patients and 223 from 953 HIV positive patients. There were 233 significant faecal isolates, 22 from 115 HIV negative patients and 211 from 531 HIV positive patients. The most common pathogens detected in blood were Streptococcus pneumoniae and Salmonella typhimurium and in faeces Shigella flexneri, S. typhimurium and Cryptosporidium parvum. The agents causing illness in HIV positive patients in Nairobi are similar to those prevalent in the HIV negative community and the investigation of a febrile illness with or without diarrhoea in an HIV positive patient should reflect this.

AIDS-Related Opportunistic Infections↗

PubMed2001

Micromosaic immunoassays.

Immunoassays are widely used for medical diagnostics and constitute the principal method of detecting pathogenic agents and thus of diagnosing many diseases. These assays, which are most often performed in well plates, would be greatly improved by a practical method to pattern a series of antigens on a flat surface and to localize their binding to many analytes. But no obvious method exists to expose a planar surface successively to a series of antigens and analytes. Here, we present miniaturized mosaic immunoassays based on patterning lines of antigens onto a surface by means of a microfluidic network (muFN). Solutions to be analyzed are delivered by the channels of a second muFN across the pattern of antigens. Specific binding of the target antibodies with their immobilized antigens on the surface results in a mosaic of binding events that can readily be visualized in one screening using fluorescence. It is thus possible to screen solutions for antibodies in a combinatorial fashion with great economy of reagents and at a high degree of miniaturization. Such mosaic-format immunoassays are compatible with the sensitivity and reliability required for immunodiagnostic methods.

Enzyme-Linked Immunosorbent Assay↗

PubMed2004

Development of an automated sample preparation module for environmental monitoring of biowarfare agents.

An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system. The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection. Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation. Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation. Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure.

Antibodies, Bacterial↗

PubMedDate not supplied

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays.

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.

3T3 Cells↗

PubMed2003

Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration.

Covalently linked to vancomycin (Van), chemically stable and highly magnetic anisotropic FePt magnetic nanoparticles (3-4 nm) become water-soluble and capture vancomycin-resistant enterococci (VRE) and other Gram-positive bacteria at concentrations approximately 10(1) cfu/mL via polyvalent ligand-receptor interactions. When a pyramidal end of a magnet "focuses" the nanoparticles into approximately 1 mm(2) area, the bacteria can be observed by an optical microscope and further identified by electron micrograph (EM). Compared to the conventional use of magnetic particles (with the sizes of 1-5 microm) in biological separation or drug delivery, magnetic nanoparticles, combined with specific receptor-ligand interactions, promise a sensitive and rapid protocol to detect pathogens.

Alloys↗