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PubMed2025

Versatile wastewater monitoring of pathogens and antimicrobial resistance enabled by metatranscriptomics and long-read metagenomics.

Widespread interest in the development of population-wide pathogen and antimicrobial resistance (AMR) monitoring has revealed wastewater's microbial footprint as a marker of public health. Near-source wastewater remains a difficult sample type for microbiome analyses but represents a closer link to human health than the downstream products of its treatment. Few studies integrate methods for non-targeted monitoring applications, and critically, current methods cannot connect AMR genes to species, nor resolve full genomes. We address these challenges by developing a pipeline that enables untargeted metagenomics, metatranscriptomics, and novel long-read metagenomics (LRG). We achieve untargeted pathogen detection, limited by highly abundant resident species, while retaining microbial information with near-source sampling. Furthermore, LRG identifies antibiotic resistance gene-containing microbes and enables assembly of culture-independent genomes with previously unreported AMR genes. We establish an integrated approach to broadly monitor pathogens in wastewater, while demonstrating the importance of LRG to illuminate microbial AMR at the species level.

Journal Article↗

PubMed1988

[The place of 67Ga scintigraphy in the primary diagnosis and follow-up evaluation of opportunistic pneumonia in patients with AIDS].

Opportunistic pneumonias are a life-threatening complication in patients with AIDS. Early diagnosis and therapy is necessary to improve prognosis. This study was designed to assess the value of 67Ga scintigraphy in the primary detection and follow-up of these special pneumonias. 67Ga scintigraphy was performed in 40 patients: 10 normal controls and 30 HIV-positive patients with AIDS or AIDS-related complex (ARC). 67Ga scan results were compared with current chest radiographs and the results of pathogen detection. The evaluation of positive scans was based on a quantification of the pulmonary uptake, expressed as a pulmonary/soft-tissue uptake ratio. Only 8/30 patients had a normal scan, 22/30 showed diffuse (13/22) or focal (9/22) increases of pulmonary uptake. In 7/8 patients with normal scans the chest radiograph was negative as well. The one patient with negative scan but positive chest radiograph had pulmonary Kaposi's sarcoma. In 11/22 patients the 67Ga scan and chest radiograph were positive simultaneously. In the other 11/22 patients with positive scans chest radiographs were initially negative but showed pathology in 5 cases within 1-2 weeks. The reason for positive scans in most cases was an opportunistic lung infection; other forms of pneumonia were only observed in two cases. The defined uptake ratio demonstrated to be a highly sensitive parameter for monitoring pneumonia and the effects of therapy in follow-up studies. In conclusion, quantitative 67Ga scintigraphy proved to be a reliable and highly sensitive method for primary detection and follow-up of opportunistic pneumonias in patients with AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed2006

Site-related airborne biological hazard and seasonal variations in two wastewater treatment plants.

Results of a study conducted to assess the degree of airborne bacterial contamination generated by two wastewater treatment plants (WWTP) with different treatment systems and evaluate the dispersion of potential pathogens, have been described. Aerosols samples were collected in summer and winter with an agar impact sampler from several plant sites. External upwind and downwind controls were also examined. Total colony-forming counts of mesophilic and thermophilic bacteria, actinomycetes and streptomycetes, Gram-negatives, coliforms and sulfite-reducers were determined. Selective media were used in order to detect pathogenic bacteria. The lowest concentrations of mesophilic and thermophilic bacteria were 8 and 28 CFU/m(3) in plants A and B respectively, the highest >40,000 CFU/m(3) in both plants. Strains of Escherichia coli, Clostridium perfringens, Staphylococcus aureus and Enterococcus spp. were isolated in some sites of the two plants. Salmonella spp., Yersinia enterocolitica and Legionella spp. were never detected. The activities involving nebulization and mechanical aeration of wastewaters and the sewage inflows have proved to be of greatest potential risk. In both plants, we found a statistically significant dependence of bacterial contamination on the season for many of the analyzed parameters but a clear seasonal trend could not be observed.

Air↗

PubMed1990

Value of 67gallium scintigraphy in primary diagnosis and follow-up of opportunistic pneumonia in patients with AIDS.

Opportunistic pneumonias are a life-threatening complication in patients with AIDS. Early diagnosis and therapy is necessary to improve the prognosis. This study was designed to assess the value of 67gallium scintigraphy in the primary detection and follow-up of these special pneumonias. 67Gallium scintigraphy was performed in 40 patients: 10 normal controls and 30 HIV-positive patients with AIDS or AIDS-related complex (ARC). 67Gallium scan results were compared with current chest x-rays and the results of pathogen detection. The evaluation of positive scans was based on a quantification of the pulmonary uptake, expressed as a pulmonary/soft tissue uptake ratio. Only 8 of 30 patients had a normal scan, while 22 of 30 showed diffuse (13/22) or focal (9/22) increases of pulmonary uptake. In seven of eight patients with normal scans the chest radiograph was negative as well. The one patient with negative scan but positive chest radiograph had pulmonary Kaposi's sarcoma. In 11 of 22 patients, the 67gallium scan and chest x-ray were positive simultaneously. In the other 11 of 22 patients with positive scans, chest radiographs were initially negative but showed pathology in five cases within 1-2 weeks. The reason for positive scans in most cases was an opportunistic lung infection; other forms of pneumonia were observed only in two cases. The defined uptake ratio was demonstrated to be a highly sensitive parameter for monitoring pneumonia and the effects of therapy in follow-up studies. In conclusion, quantitative 67gallium scintigraphy proved to be a reliable and highly sensitive method for primary detection and follow-up of opportunistic pneumonias in patients with AIDS.

AIDS-Related Complex↗

PubMed2006

Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli 0157:H7 and Listeria monocytogenes.

Escherichia coli 0157:H7 and Listeria monocytogenes are the two most important food-borne human pathogens. To develop a single, rapid and sensitive PCR based test for simultaneous detection of both the organisms, fliCh7 and iap gene specific primers were used respectively for E. coli 0157:H7 and L. monocytogenes. Initially, with equal quantities of purified genomic DNAs of these organisms a multiplex PCR reaction was standardized to yield uniform amplification of both targets. Although, this assay detected E. coli 0157:H7 with high sensitivity, it failed to pick up L. monocytogenes after several hours of enrichment in broth medium initially spiked with equal numbers of live cells. This was found to be due to unequal growth of these organisms leading to disparity in the amount of template DNAs represented in the DNA preparation applied for conventional multiplex PCR amplification. To circumvent this, we have developed a modified method of enrichment and harvesting leading to highly sensitive and rapid single reaction PCR detection of both pathogens. We have also successfully developed two novel multiplex PCR formats for the generation of uniform PCR signals. Some of these methods might find broader application for the simultaneous detection of different combinations of multiple pathogens.

Bacterial Proteins↗

PubMed2005

Epidemiology of community-acquired pneumonia in adult patients at the dawn of the 21st century: a prospective study on the Mediterranean coast of Spain.

This study presents data from a prospective study of adult patients with community-acquired pneumonia (CAP). Of 493 patients included in the study, 223 (45.2%) were aged > or = 65 years, and 265 (53.7%) had one or more underlying diseases, mostly chronic obstructive pulmonary disease, diabetes mellitus or dementia. In total, 281 microorganisms were identified in 250 (50.7%) patients, with two or more pathogens detected in 28 (5.7%) cases. Microbial diagnosis varied according to age, severity, co-morbidity and site-of-care, but there was much overlap among groups. Streptococcus pneumoniae was the single most prevalent organism in outpatients, patients admitted to hospital, and patients who died, either as a single pathogen or combined with another organism. Infections caused by 'atypical' pathogens were seen across all groups, including the elderly and patients with co-morbidities. Mortality varied according to the pneumonia severity index (PSI) of the pneumonia patient outcomes research team. Shock (OR 34.48), an age of > 65 years (OR 25) and altered mental status (OR 9.92) were factors associated independently with 30-day mortality. Key findings from this study were the advanced age of the population with CAP, and the high prevalence of dementia as an underlying disease. The study also revealed that microbiological diagnosis of CAP remains problematic. Although certain epidemiological features may help to predict the microbial aetiology, the overlap among groups reduces the usefulness of this information in guiding therapeutic decisions. Greater effort should be made to improve identification methods for microbial pathogens causing CAP.

Adolescent↗

PubMed1989

Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction.

We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

Animals↗

PubMed2003

[Clinical efficacy of oral clarithromycin monotherapy in patients with mild or moderate community-acquired pneumonia].

Clarithromycin (CAM) is a new macrolide antibiotic which is active against a wide range of organisms responsible for community-acquired pneumonia (CAP) and has superior pharmacokinetics and tolerance compared to erythromycin. In this study, we evaluated the clinical efficacy and antimicrobial activity of CAM in the empirical treatment of patients with CAP. CAM (200 mg given twice daily for 2 weeks) was orally administered to 26 patients with mild or moderate suspected atypical pneumonia, including 15 patients in whom treatment with beta-lactam antibiotics was largely ineffective. Causative pathogens were identified on the basis of quantitative sputum cultures, blood cultures, and routine serological testings; M. pneumoniae was most commonly observed in patients with CAP (38.5%; 10/26), followed by H. influenzae (11.5%; 3/26), C. pneumoniae (3.8%; 1/26), and S. constellatus (3.8%; 1/26). Penicillin-resistant, or penicillin-susceptible Streptococcus pneumoniae were isolated from 1 patient (3.8%) and 2 patients (7.7%), respectively out of 26 patients with CAP. There were no detectable pathogens in 8 of 26 patients. The treatment of CAM resulted in complete resolution of all signs and symptoms of pneumonia in all the patients and was not accompanied with any adverse events. The overall incidence of laboratory abnormalities was not detectable in the patients evaluated. Although it is important to make differential diagnosis of atypical from bacterial pneumonia in designing therapeutic strategy, it is often difficult to make an appropriate diagnosis in patients with CAP. Because of diagnostic difficulties, CAM with a broad antimicrobial spectrum is recommended as the first-line drug for the treatment of lower respiratory infections, particularly in patients with suspected atypical pneumonia.

Administration, Oral↗

PubMed1994

Light-generated oligonucleotide arrays for rapid DNA sequence analysis.

In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition.

Base Sequence↗

PubMed1990

Clinical and parasitologic aspects of cryptosporidiosis in nonhuman primates.

Eighty-one cases of acute cryptosporidiosis were diagnosed among 157 (52%) infant primates, predominantly Macaca nemestrina, housed in the nursery unit of the Washington Regional Primate Research Center. The mean age at onset of oocyst passage was 38 +/- 25 days. The outbreak was confined to the nursery and no cases were detected among juvenile or adult primates housed in other rooms within the colony. All but one animal manifested symptoms of enteric infection, including severe diarrhea and dehydration. Infected animals excreted oocysts for a mean of 36 days (range 7-78 days). No reinfections occurred. Cryptosporidium was the second most common enteric pathogen detected in the population, after Campylobacter jejuni. The risk of infection was related to the length of time the animal was housed in the nursery and to social interaction with other monkeys. These findings are relevant to the understanding of the epidemiology of cryptosporidiosis among human infants and children in environments with close social interactions and minimal learned personal hygiene practices.

Animals↗

PubMed2003

Clinical findings among 62 Thais with cholangiocarcinoma.

Cholangiocarcinoma, a malignancy of the biliary duct system, tends to grow slowly and to infiltrate the walls of the ducts, dissecting along tissue planes and leading to biliary tract obstruction. In a retrospective case review, 62 cases diagnosed with cholangiocarcinoma between January 1997 and December 2001 at the King Chulalongkorn Memorial Hospital in Bangkok were studied. The most commonly observed clinical symptoms and signs were fever (96.8%), abdominal pain (87.1%) and malaise or weakness (83.9%). Fifty-one of the 62 tumours detected were distal extrahepatic (82.3%), seven perihilar (11.3%) and four intrahepatic (6.7%). There was a significant increase in serum bilirubin and marked elevation of serum alkaline phosphatase. Almost half (45.2%) of the patients presented with other pathological conditions, mostly cholangitis (19 cases) resulting from acute biliary tract obstruction and ascites (13 cases). The pathogens detected were similar in cases of cholangitis alone and cholangiocarcinoma. The major risk factor for this cancer in Thailand is believed to be exposure to liver fluke in insufficiently cooked traditional foods. As it is still endemic in Thailand, continuous prevention and surveillance of this public health problem are necessary.

Adult↗

PubMed1992

Reverse transcription and polymerase chain reaction amplification of rRNA for detection of Helicobacter species.

Sequence data on Helicobacter pylori 16S rRNA were used to select two 22-base oligonucleotide primers for use in a polymerase chain reaction (PCR) for detection of H. pylori. H. pylori cells were treated with lysis buffer, boiled, and chloroform extracted. Reverse transcription of rRNA was followed by PCR amplification (RT-PCR) of the synthesized cDNA and 16S rRNA gene. The amplified PCR products were analyzed by agarose gel electrophoresis and Southern blotting. Using ethidium bromide-stained agarose gels, we were able to detect the expected 500-bp DNA fragment from as few as two H. pylori organisms per reaction. The specificity of the RT-PCR assay was tested with 27 clinical isolates and related reference strains; although the number of bacterial cells used per reaction was 10(5)-fold greater than the number of H. pylori organisms used, amplification was detected only with bacteria in the same genus, H. cinaedi and H. mustelae. Ten H. pylori organisms per biopsy specimen were detected on agarose gels when organisms were added to samples prepared from a processed colon biopsy sample. RT-PCR results were consistent with urea breath test and culture results in 14 of 15 gastric biopsy specimens; the specificity was 100%. RT-PCR of rRNA from H. pylori increased the sensitivity of pathogen detection at least 25- to 50-fold compared with that of previous PCR assays. This low level of detection by RT-PCR assay may prove to be well suited for verifying eradication following therapy.

Base Sequence↗

PubMed2001

Prevalence of viral, bacterial and parasitic enteropathogens among young children with acute diarrhoea in Jeddah, Saudi Arabia.

The prevalence of viral, bacterial and parasitic pathogens among children of Jeddah, Saudi Arabia, was investigated. During December 1995-October 1996, 576 faecal samples were collected from children (0-5 year(s) old) suffering from acute diarrhoea and attending hospitals and outpatient clinics in Jeddah. One or more enteropathogen(s) were identified in 45.6% of the stool specimens. Mixed infections were detected in 12.2% of the diarrhoeal cases. Rotavirus was detected in 34.6% of the specimens of the hospitalized patients and in 5.9% of the specimens of the outpatients. Fifty-one percent of the rotavirus-positive specimens were long electropherotype, 26% were short electropherotype, and 23% could not be electropherotyped specifically. Among those of the long electropherotype, there were six patterns; and of the short electropherotypes, there were four patterns. Serotyping of these specimens revealed a distribution of 39.6%, 4.2%, 6.3%, and 15.6% for rotavirus serotype 1, 2, 3, and 4 respectively. Mixed serotypes were found in 3.1%, and 31.3% of the specimens were untypeable. Other aetiologic agents recognized included Escherichia coli (13%), of which 3.8% were enteropathogenic E. coli (EPEC) and 1.9% enterohaemorrhagic E. coli. Among the E. coli (EPEC) serotypes, O111:K58:B4, O55:K59:B11, and 0127:K63:B8 were found in 31.8%, 18.2%, and 13.6% of the cases respectively. Serotype 026:K60:B6, 0124:K72:B17, and 0112:K66:B11 each was found in 9.1% of the EPEC cases. 0128:K67:B12 and 0125:K70:B13 each was found in one case only. Other detected pathogens were: Klebsiella pneumoniae (4%), Giardia lamblia (3.1%), Salmonella sp. (3%), Shigella flexneri (2.6%), Entamoeba histolytica (2.2%), Trichuris trichiura, Hymenolepis nana, and Ascaris lumbricoides (0.7% each), and Candida albicans (0.5%). Based on the results of this study, it is concluded that the high prevalence of the various enteropathogens among young children is a significant public health problem.

Child, Preschool↗

PubMed1996

Autoimmune manifestations in the transforming growth factor-beta 1 knockout mouse.

Targeted disruption of the transforming growth factor-beta 1 (TGF-beta 1) gene in mice results in the development of a massive multifocal inflammatory disease in many tissues. Because no detectable pathogen was identified, we examined whether autoimmune mechanisms played a role in initiating or maintaining the inflammatory disease. The serum of TGF-beta 1 knockout mice contained elevated titers of antibodies to nuclear antigens (ssDNA, dsDNA, Sm, and RNP) as well as reactivity against the 16/6 idiotype (16/6 Id). In addition, Ig deposits were detected in renal glomeruli of TGF- beta 1 knockout mice. Transplantation of TGF-beta 1 knockout hematopoietic cells into normal irradiated recipients resulted in a similar profile of autoantibody production as well as in the induction of inflammatory lesions. Our results describe autoimmune activity that ensues when the TGF-beta 1 cytokine is absent.

Animals↗

PubMed2005

[Bacterial detection leading to pathogen inactivation].

Bacterial contamination of blood components remains the highest infectious risk in blood transfusion, the risk is particularly high when it affects platelet concentrates (PC). In France, the residual risk of transfusion reaction due to bacterial contamination of PC has been decreasing slowly since 1994 but for all severity 1 case occurs with about 25,000 distributed PC and one death occurs with 200,000 distributed units. This reduction of the risk may be due to the measures which were implemented during the last 10 years in order to prevent contamination during donation. Improving strategies for reducing the risks of bacterial contamination is one of the priorities of the French National Blood Transfusion Service (l'Etablissement Français du sang - EFS). The main target remains PC. Bacterial detection or pathogens inactivation are now available and are able to reduce (for detection) or prevent (for inactivation) the occurrence of reaction due to bacterial contamination of PC. Up to now, the choice is in favour of bacterial detection. A national study was carried out in seven regional EFS at the end of 2004. It aims at confirming the feasibility of a systematic bacterial screening of PC before their delivery. The first conclusions show that this screening can be implemented with acceptable modifications in term of platelets availability. We can expect in a next future that new pathogens reduction technique and/or new detection systems will be available, certainly more efficient to prevent reaction due to bacterial contamination. Implementation of actual detection methods is probably a temporary solution.

Bacteriological Techniques↗

PubMed2000

Advances in Understanding Plant Viruses and Virus Diseases.

▪ Abstract Plant viruses have had an impact on the science of virology and on plant pathology ever since the virus concept was discovered with Tobacco mosaic virus at the end of the nineteenth century. In this review, we highlight those discoveries. We have divided plant virus research into a "Classical Discovery Period" from 1883-1951 in which the findings were very descriptive; an "Early Molecular Era" from 1952 to about 1983, in which information was developed that described further properties of the viruses, aided by the development of a number of salient techniques; and the "Recent Period" from 1983 to the present, when techniques have been developed to modify plant virus genomes, to detect nonstructural gene products, to determine the functions of viral gene products, and to transform plants to elicit novel forms of resistance to viral diseases. In this period, plant virology has played a significant role in formulating an understanding of the mechanisms of gene silencing and recombination, plasmodesmatal function, systemic acquired resistance, and in developing methods for pathogen detection. We also attempt to predict the direction plant virology will take in the future.

genetic engineering↗

PubMed2006

Mutations in the Wilms' tumor 1 gene cause isolated steroid resistant nephrotic syndrome and occur in exons 8 and 9.

Primary steroid-resistant nephrotic syndrome (SRNS) is characterized by childhood onset of proteinuria and progression to end-stage renal disease. Approximately 10-25% of familial and sporadic cases are caused by mutations in NPHS2 (podocin). Mutations in exons 8 and 9 of the WT1 gene have been found in patients with isolated SRNS and in SRNS associated with Wilms' tumor (WT) or urogenital malformations. However, no large studies have been performed to date to examine whether WT1 mutations in isolated SRNS are restricted to exons 8 and 9. To address this question, we screened a worldwide cohort of 164 cases of sporadic SRNS for mutations in all 10 exons of the WT1 gene by multiplex capillary heteroduplex analysis and direct sequencing. NPHS2 mutations had been excluded by direct sequencing. Fifteen patients exhibited seven different mutations exclusively in exons 8 and 9 of WT1. Although it is possible that pathogenic mutations of WT1 may also reside in the introns, regions of the gene that were not able to be screened in this study, these data together with our previous results (Ruf et al.: Kidney Int 66: 564-570, 2004) indicate that screening of WT1 exons 8 and 9 in patients with sporadic SRNS is sufficient to detect pathogenic WT1 mutations and may open inroads into differential therapy of SRNS.

Amino Acid Sequence↗

PubMed1994

Prevalence of periodontal pathogens with varying metabolic control of diabetes mellitus.

The purpose of this study was to determine the prevalence of 5 periodontal pathogens in individuals with diabetes mellitus. Subjects (n = 107) 20-70 years of age with type 1 (n = 60) or 2 (n = 47) diabetes mellitus were studied for the occurrence of the periodontal pathogens A. actinomycetemcomitans, F. nucleatum, E. corrodens, P. gingivalis and P. intermedia. Subgingival plaque was sampled in each subject from a single site exhibiting the greatest inflammation. The evaluation of selected periodontal bacterial pathogens was based on an immunoassay utilizing bacterial specific monoclonal antibodies. 35% of the sites harbored P. gingivalis, 28% F. nucleatum and 21% E. corrodens. A. actinomycetemcomitans and P. intermedia were found in less than 10% of the sites. Subjects for whom the probing depth at the sampled site was > or = 4 mm were more often found to have detectable pathogens than those with a probing depth < or = 3 mm. Diabetic factors such as duration, type and metabolic control of the disease had no statistically significant effect on the prevalence of these bacteria.

Adult↗