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PubMed1995

Using oligonucleotide probe arrays to access genetic diversity.

As the Human Genome Project and related efforts identify and determine the DNA sequences of human genes, it is important that highly reliable and efficient mechanisms are found to access individual genetic variation. It is only through a greater understanding of genetic diversity that the true benefit of the Human Genome Project will be realized. One approach, hybridization to high-density arrays of oligonucleotides, is a fast and effective means of accessing this genetic variation. Light-directed chemical synthesis has been used to generate miniaturized, high-density arrays of oligonucleotide probes. Application-specific oligonucleotide probe array designs have been developed for the rapid screening of characterized genes. Dedicated instrumentation and software have been developed for array hybridization, fluorescence detection and data acquisition and analysis. In a specific and challenging application, oligonucleotide probe arrays have been used to screen the reverse transcriptase and protease genes of the highly polymorphic HIV-1 genome to explore genetic diversity and detect mutations conferring resistance to antiviral drugs. Results from this application strongly suggest that oligonucleotide probe arrays will be a powerful tool for rapid investigations in sequence checking, pathogen detection, expression monitoring and DNA molecular recognition.

Base Sequence↗

PubMed1987

Cryptosporidiosis in well-nourished and malnourished children.

During a 5-month period, 513 stool samples submitted to the enteric laboratory at the University Hospital of the West Indies were examined for Cryptosporidium. Oocysts were detected in 4.9% of all stools, 7.3% of diarrhoeal stools, 19.5% of stools from malnourished children and 23.7% of stools from malnourished children with diarrhoea. Cryptosporidium was the sole pathogen detected in all 25 positive stools, and was the second most frequent enteric isolate. All cases of cryptosporidiosis occurred in children less than 2.5 years of age. All 15 malnourished children were admitted to hospital where they presented with dehydration (87%), vomiting (93%), fever (100%) and diarrhoea which lasted an average of 15.3 days. Two of these children died. In contrast, dehydration (20%), vomiting (40%) and fever (50%) were less common and diarrhoea less protracted in well-nourished children, four of whom were admitted to hospital. This preliminary report suggests that cryptosporidial gastroenteritis presents with increased frequency and severity in malnourished compared with well-nourished Jamaican children.

Child, Preschool↗

PubMed2024

Beyond water and soil: Air emerges as a major reservoir of human pathogens.

Assessing the risk of human pathogens in the environment is crucial for controlling the spread of diseases and safeguarding human health. However, conducting a thorough assessment of low-abundance pathogens in highly complex environmental microbial communities remains challenging. This study compiled a comprehensive catalog of 247 human-pathogenic bacterial taxa from global biosafety agencies and identified more than 78 million genome-specific markers (GSMs) from their 17,470 sequenced genomes. Subsequently, we analyzed these pathogens' types, abundance, and diversity within 474 shotgun metagenomic sequences obtained from diverse environmental sources. The results revealed that among the four habitats studied (air, water, soil, and sediment), the detection rate, diversity, and abundance of detectable pathogens in the air all exceeded those in the other three habitats. Air, sediment, and water environments exhibited identical dominant taxa, indicating that these human pathogens may have unique environmental vectors for their transmission or survival. Furthermore, we observed the impact of human activities on the environmental risk posed by these pathogens, where greater amounts of human activities significantly increased the abundance of human pathogenic bacteria, especially in water and air. These findings have remarkable implications for the environmental risk assessment of human pathogens, providing valuable insights into their presence and distribution across different habitats.

Humans↗

PubMed2026

The Next Step: The Role of Metagenomic Next-Generation Sequencing in Microbial Detection of Culture-Negative Cardiovascular Infections.

Cardiovascular infections, including those that involve native and prosthetic heart valves, implantable cardiac devices, mechanical circulatory assist devices, and vascular grafts, are associated with significant morbidity and mortality risks. Optimal management of these complex infections requires pathogen-directed antimicrobial therapy. However, standard culture-based methods often fail to identify causative organisms due to prior antimicrobial use, infections due to fastidious organisms, or biofilm-associated infections. Emerging evidence suggests that microbial cell-free DNA (mcfDNA) and metagenomic testing can enhance pathogen detection, particularly in culture-negative cases. However, their results require careful clinical interpretation, often necessitating input from infectious diseases specialists. In this review, we examine published evidence regarding metagenomic testing for cardiovascular infections and its impact on patient care. We propose a framework for microbiological adjudication of mcfDNA results, introduce standardized definitions for clinical impact assessment, and provide guidance on integrating mcfDNA testing into diagnostic evaluation of patients with culture-negative cardiovascular infections.

Humans↗

PubMed1995

Evaluation of periodontal treatments using controlled-release tetracycline fibers: microbiological response.

In a 12-month multi-center study of 116 adult periodontitis subjects, six putative periodontal pathogens were monitored by DNA probe methods in a subset of 31 subjects. Monitored species included Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), Eikenella corrodens (Ec), Campylobacter rectus (Cr), and Actinobacillus actinomycetemcomitans (Aa) with an average detection limit of 1.8 x 10(4) bacterial colony forming units/sample. The microbiological response to four periodontal treatments was studied, one treatment in each quadrant; scaling and root planing (S), scaling and root planing with tetracycline (TC) fiber (SF), a single application of TC fiber (F) and two serial applications of TC fiber (FF). Generally two sites were sampled in each quadrant, however, in some quadrants only one site was selected. These treatments were evaluated at baseline; immediately following therapy; and post-treatment at 1, 3, 6, and 12 months. The study was conducted with a split-mouth design with no maintenance therapy over a 12-month period. At baseline, 70.8% of sites had detectable Fn; 42.9% Pg; 63.5% Pi; 29.7% Ec; 28.3% Cr; and 5.5% Aa. No significant differences were seen in baseline proportions of these species between centers. Numbers and proportions of detectable pathogens (with the exception of Pg) exhibited a triphasic temporal response: a precipitous initial decrease immediately following therapy; a rise in proportions in the 1- to 3-month post-therapy period; and a spontaneous decline in the absence of therapy over the 3- to 12-month period.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1989

Diagnostic studies of nosocomial diarrhea in children: assessing their use and value.

During a 17-month period (01/11/85-05/31/86) 225 cases of nosocomial diarrhea were identified in a children's hospital. Diarrhea was considered to be nosocomial if it began at least 72 hours after the patient's hospital admission or within 3 days after discharge. One or more routine diagnostic studies for identification of a pathogen were performed in 195 (87%) cases. The most commonly performed test was the bacterial stool culture. None of these samples yielded a bacterial pathogen. The only pathogens detected by routine laboratory studies were rotavirus (61/137 [45%] samples were positive for rotavirus by ELISA) and Clostridium difficile (9/54 [17%] positive for toxin). Of the patients whose tests were positive for rotavirus 56 were younger than 2 years of age, and all were identified in the winter and spring. When multiple stool samples were tested by the diagnostic laboratory, rotavirus was identified in an additional 14 patients whose initial stool samples were negative for rotavirus. All patients whose tests were positive for C. difficile toxin had received antibiotics within the previous 3 months. Ova/parasites were not detected in 53 of the tested stools. We also identified enteric adenovirus in six patients. Viruses were identified in 95 (42%) of the 225 cases of nosocomial gastroenteritis. Nosocomial diarrhea is common in a children's hospital. Rotavirus is the most commonly identified pathogen. Rotavirus testing is valuable in children with nosocomial diarrhea who are younger than 2 years of age, especially in the winter and spring. Multiple samples may be necessary to identify rotavirus. C. difficile toxin assay should be considered for patients who are receiving or who have received antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross Infection↗

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↗

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↗

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↗

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↗

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↗

PubMed2026

Diagnostic value of plasma cell-free DNA metagenomic next-generation sequencing in patients with suspected infections and exploration of clinical scenarios-a retrospective study from a single center.

BACKGROUND: Plasma cell-free DNA metagenomic next-generation sequencing (mNGS) is a non-invasive comprehensive method for the etiological diagnosis of various infectious diseases. However, research on the early diagnosis and real-world clinical impact of plasma mNGS in patients with suspected infection are still limited. MATERIALS AND METHODS: This study retrospectively included 140 patients with suspected infections who underwent early plasma mNGS and conventional culture testing. Referring to the clinical diagnosis of infectious diseases, the diagnostic performance of plasma mNGS and culture tests was compared, and the application scenarios and clinical effects of plasma mNGS were evaluated. RESULTS: The positive rate of plasma mNGS was significantly higher than that of culture methods (55.71% vs 25.10%, p < 0.001) and blood cultures (55.71% vs 12.86%, p < 0.001). Regarding clinical diagnosis, the sensitivity of plasma mNGS was significantly higher than that of culture (58.27% vs 37.80%, p = 0.002). The combination of mNGS and culture achieved a higher detection sensitivity (69.29%), especially in patients with multi-site co-infections (73.68%) and blood infections (73.17%). Plasma mNGS demonstrated higher sensitivity in patients with procalcitonin (PCT) index > 5 ng/ml or human neutrophil lipocalin (HNL) index > 200 ng/ml. In terms of treatment, a total of 69 patients (54.33%) benefited from plasma mNGS. CONCLUSION: This study highlights the significant improvement in pathogen detection performance by combining conventional culture with plasma mNGS detection, especially in patients with multi-site co-infections and blood infections. Early use of plasma mNGS as an adjunct to culture can better guide clinicians to initiate appropriate anti-infective therapy.

Humans↗

PubMed1985

Pathogenic properties of Campylobacter jejuni: assay and correlation with clinical manifestations.

The pathogenic properties of 20 strains of Campylobacter jejuni isolated from persons with clearly defined clinical manifestations were determined. Cell-free broth filtrates were examined for (i) enterotoxin production by Chinese hamster tissue culture assay and an enzyme-linked immunosorbent assay (ELISA) employing GM1 ganglioside and affinity-purified antiserum to Escherichia coli heat-labile toxin, (ii) cytotoxin production by Vero and HeLa cell tissue culture lines, and (iii) their ability to cause fluid secretion in rat ligated ileal loops. Viable bacteria were examined for invasive properties by an ELISA with the immunoglobulin fraction of antiserum to Formalin-killed bacteria of an invasive strain, and by their effect on fluid secretion and morphology in rat ligated ileal loops. None of the eight isolates obtained from asymptomatic carriers had any detectable pathogenic properties. All six strains isolated from persons with bloody invasive-type diarrhea elaborated a cytotoxin; their viable bacteria had high titers in the ELISA for invasive properties and caused fluid secretion in ligated ileal loops, although consistent morphologic abnormalities and evidence of mucosal invasion, examined by immunofluorescence techniques, were not detected. All six strains isolated from persons with watery secretory-type diarrhea produced an enterotoxin, one elaborated a cytotoxin, and broth filtrates of all strains caused fluid secretion in ligated ileal loops; viable bacteria had low titers in the ELISA for invasive properties and evoked fluid secretion in ligated loops by means of enterotoxin production. These observations show (i) that a correlation exists between the pathogenic properties of the infective C. jejuni strain and gastrointestinal manifestations in the infected host, and (ii) that these pathogenic properties can be identified by in vitro assays, including ELISAs.

Animals↗

PubMed1991

[Clinicopathological study of methicillin-resistant Staphylococcus aureus detected by pulmonary microbial culture in autopsied cases].

Microbial culture of lung specimens from 569 autopsied cases from 1986 to 1989 revealed methicillin-resistant Staphylococcus aureus (MRSA) in 28 cases, which were subsequently analyzed clinicopathologically. The number of MRSA positive cases has markedly increased in recent years (2 cases in 1986, 2 in 1987, 6 in 1988, 18 in 1989). The most frequent underlying disease was neoplasm, which was seen in 17 cases. Of non-neoplastic diseases, liver cirrhosis and diffuse panbronchiolitis were prevalent. Twenty-four cases had received a course of antibiotic therapy. Antibiotics frequently administered were third-generation Cephem and Imipenem/cilastatin sodium (used in 20 cases). Antibiotics o which MRSA was sensitive were administered in only one case (minocycline). Sputum culture was performed in only 10 cases, 5 of which were MRSA positive. MRSA had acquired resistance to fosfomycin and ofloxacin. Histological examination revealed complication by pneumonia in 19 cases. In 7 of these 19 cases, MRSA was the only pathogen detected. Pulmonary MRSA infection detected at autopsy is frequently seen in patients with terminal stage cancer, but it is frequently not diagnosed and is undertreated. This may be a factor responsible for the recent marked increase in the proportion of MRSA in pathogens causing infection within medical institutions.

Cross Infection↗

PubMed2026

Seqwin: ultrafast identification of signature sequences in microbial genomes.

MOTIVATION: Polymerase chain reaction (PCR) enables rapid, cost-effective diagnostics but requires prior identification of genomic regions that allow sensitive and specific detection of target microbial groups, herein referred to as microbial signature sequences. We introduce Seqwin, an open-source framework designed to automate microbial genome signature discovery. Tens of thousands of microbial genomes are now available for a single species, limiting the application of existing manual and automated approaches for identifying signatures. Modern approaches that are capable of leveraging all available microbial genomes will ensure sensitive and accurate DNA signature identification and enable robust pathogen detection for clinical, environmental, and public health applications. RESULTS: Seqwin builds weighted pan-genome minimizer graphs and uses a traversal algorithm to identify signature sequences that occur frequently in target genomes but remain rare in non-targets. Unlike earlier tools that depend on strict presence or absence of sequences, Seqwin accommodates natural sequence variation and scales to very large genome collections. When applied to genomes from C. difficile, M. tuberculosis, and S. enterica, Seqwin recovered more high-quality signatures than alternative methods with lower computational burden. Seqwin's analysis of nearly 15 000 S. enterica genomes yielded over 200 candidate signatures in three minutes. Seqwin provides an open-source solution for the long-standing need for scalable microbial signature discovery and diagnostic assay design. AVAILABILITY AND IMPLEMENTATION: Seqwin is available on GitHub (https://github.com/treangenlab/Seqwin) and can be installed via Bioconda (https://bioconda.github.io/recipes/seqwin/README.html). Benchmarking datasets, outputs, and scripts are available on Zenodo (https://doi.org/10.5281/zenodo.19874011).

Software↗

PubMed1989

Chronic colitis after Aeromonas infection.

Three patients with an acute colitis in which the only pathogen detected was either Aeromonas hydrophila or A sobria progressed to a chronic phase after the infection had been eliminated by antibiotic treatment in two and had resolved spontaneously in the third. The final diagnosis in each case was ulcerative colitis. Two of the patients have responded to anti-inflammatory medication but one has required panproctocolectomy. The sequence of symptoms and observations in these cases, as well as in others from the literature involving more familiar pathogens, suggests that bacterial infection may contribute to the development of chronic colitis. This supposition could be tested by extending the follow up of patients with acute infective colitis in a prospective multicentre trial.

Adult↗

PubMed1990

New molecular techniques for microbial epidemiology and the diagnosis of infectious diseases.

A major aim of clinical microbiologists over the last century has been to demonstrate the presence of pathogenic microorganisms in clinical or pathologic samples associated with infectious diseases. With the development of molecular genetics over the last two decades, new technologies have become available that allow more sensitive and specific determinations to be made in shorter periods. Two considerable benefits have accrued: Epidemiologists are now capable of judging clonality among various clinical isolates more powerfully, permitting added accuracy in the evaluation of the epidemic spread of microbes, and clinicians have gained enormous ability to diagnose previously difficult-to-detect pathogens.

Bacteria↗