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PubMedDate not supplied

[Prevalence of enteropathogens in children with liquid diarrhea].

Between january 1 and october 31, 1991 148 samples of feces corresponding to the same number of children with acute diarrhea, aged 0-2 years, admitted to Hospital O'Horan of the Secretariat of Health in the city of Merida, Yucatan, Mexico, were studied. Stools from 105 (70.9%) patients were described as watery diarrhea; from these samples at least one enteric pathogen was detected in 49 (46.6%): a single pathogen was isolated in 37 (35.2%), and mixed infections were observed in 12 (11.4%). We isolated enterotoxigenic Escherichia coli in 18 (17.1%) samples; a predominance of strains producing only ST (11.4%) was observed. Rotavirus was detected in 14.3%, Shigella spp in 8.6%, Salmonella spp in 5.7%, Giardia lamblia in 4.8%, Campylobacter jejuni in 3.8%, enteroinvasive E. coli in 2.8%, Entamoeba histolytica in 2.8% and adenovirus in 1.9%. From watery samples, 80 (76.2%) were negative for fecal blood, and these were most frequently associated with enterotoxigenic E. coli (17.5%) and rotavirus (12.5%). E. coli LT+ and rotavirus association was observed in two patients, and E. coli ST+ and adenovirus association was observed in another one.

Adenoviridae Infections↗

PubMed1998

HIV enteropathy: comparative morphometry of the jejunal mucosa of HIV infected patients resident in the United Kingdom and Uganda.

AIMS: To compare jejunal mucosal morphometry in HIV infected patients resident in London and Uganda. PATIENTS: Twenty HIV positive patients from London and 16 from Uganda were studied, and compared with HIV negative control subjects from both sites. METHODS: Stools and biopsy specimens were examined for enteropathogens. Surface area to volume (S:V) ratio was estimated morphometrically, mean crypt length of jejunal biopsy specimens was measured, and HIV infected cells detected immunohistochemically were quantified. RESULTS: Enteric pathogens were detected in none of the London patients, and in three Ugandan patients. S:V ratio was lower, and mean crypt length higher, in the specimens of London patients than in normal subjects, but there was no difference in S:V ratio or mean crypt length between Ugandan patients and controls. A negative correlation was present between S:V ratio and mean crypt length in all biopsy specimens analysed. HIV infected cells were detected only in lamina propria. CONCLUSION: Infection of cells in the lamina propria of the jejunum with HIV stimulates crypt cell proliferation, and a fall in villous surface area. The mucosal response to HIV is masked by other pathogens in the African environment.

Adult↗

PubMed1995

Single-step PCR for detection of Brucella spp. from blood and milk of infected animals.

A versatile method for the extraction of Brucella DNA and PCR are presented as reliable tools for the detection of Brucella spp. from body fluids of infected animals. Two oligonucleotides homologous to regions of the gene encoding for an outer membrane protein (omp-2) were designed to detect the pathogen from milk and/or blood of infected goats, bovines, and human patients. The sensitivity of our test and its ability to detect the pathogen in samples from the field reveal a promising advance in the diagnosis of brucellosis in animals and humans.

Animals↗

PubMed2000

[Development and testing of polymerase chain reaction method for detection of meningococcal infection pathogen].

A system of primers has been developed for the diagnosis of meningococcal infection has been developed on the basis of the polymerase chain reaction (PCR). Specificity and sensitivity of detection of N. meningitidis has been evaluated in analysis of the model clinical specimens. The proposed test system was tried in detection of N. meningitidis in nasopharyngeal discharge specimens from patients with acute nasopharyngitis and normal subjects from the focus of meningococcal infection.

Humans↗

PubMed2001

Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections.

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.

DNA, Fungal↗

PubMed2006

[A study to assess the microbial contamination of Mya arenaria clams from the north shore of the St Lawrence River estuary, (Québec, Canada)].

The aims of the present study were to assess the microbial quality of Mya arenaria clams from the north shore of the St. Lawrence River estuary and to validate various microbial indicator microorganisms of bivalve mollusks contamination. Clams were collected from nine sites, including four harvesting sites closed by virtue of the Canadian Shellfish Sanitation Program (CSSP). Six contamination indicators (fecal coliforms, somatic coliphages, F-specific coliphages, fecal streptococci, Clostridium perfringens, and Escherichia coli) and four pathogens (Campylobacter sp., Cryptosporidium parvum, Giardia sp., and Salmonella sp.) were identified in the clams. Indicators sensibility, specificity and predictive values with respect to the presence of pathogens were calculated. Pathogenic microorganisms detection frequency in clams was important (92%). Globally, pathogens tend to be less frequently detected in opened harvesting sites (p = 0.086). Although the assessed indicators were not perfect, when F-specific coliphages are associated with E. coli or fecal coliforms, a good sensibility (62%-64%) and good positive predictive value (88%) with respect to the investigated pathogens are obtained.

Animals↗

PubMed2002

Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens.

We describe a novel adaptation of the TaqMan PCR assay which potentially allows for highly sensitive detection of any eubacterial species with simultaneous species identification. Our system relies on a unique multiprobe design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair and is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step effectively decontaminates or removes background DNA. The TaqMan system described reliabAly detected 14 common bacterial species with a detection limit of 50 fg. Further, highly sensitive and specific pathogen detection was demonstrated with a prototype species-specific probe designed to detect Staphylococcus aureus. This assay has broad potential in the clinical arena for rapid and specific diagnosis of infectious diseases.

Bacteria↗

PubMed1996

The use and abuse of routine stool microbiology: a College of American Pathologists Q-probes study of 601 institutions.

OBJECTIVE: To examine the efficiency with which physicians use routine stool microbiology tests. DESIGN: Questionnaire and structured review of 100 consecutive stool bacteriology and parasitology examinations at each participating institution. SETTING: Six hundred one institutions enrolled in the College of American Pathologists Q-probes Program. RESULTS: Of 59500 bacteriology specimens, 3808 (6.4%) contained a pathogen. The vast majority (99%) of bacterial pathogens were detected in either the first or second specimen submitted. Almost 40% of inpatient specimens were collected after the third day of hospitalization, but only 0.6% of these specimens were positive for enteric pathogens that had not been previously recovered. More than half of the laboratories reported having no limits on the number of bacteriology specimens per patient that could be submitted for testing, and fewer than 8% of laboratories rejected specimens from inpatients after a certain number of days in the hospital. The frequency with which laboratories performed tests for Clostridium difficile varied widely. Of 58500 parasitology specimens, 1463 (2.5%) contained a pathogen; 97.6% of pathogens were detected by the second stool specimen, and 99.8% were detected by the third specimen. Only 0.7% of specimens from inpatients hospitalized more than 4 days contained a new pathogen. CONCLUSIONS: We recommend that no more than two bacteriology specimens and no more that two or three parasitology specimens be processed per patient without consultation. Standard stool examination for a bacterial pathogens has a low yield and should not be performed after 3 days of hospitalization. Likewise, parasitology examinations should not be performed after 4 days of hospitalization.

Clostridioides difficile↗

PubMed2005

Use of artificial neural networks to accurately identify Cryptosporidium oocyst and Giardia cyst images.

Cryptosporidium parvum and Giardia lamblia are protozoa capable of causing gastrointestinal diseases. Currently, these organisms are identified using immunofluorescent antibody (IFA)-based microscopy, and identification requires trained individuals for final confirmation. Since artificial neural networks (ANN) can provide an automated means of identification, thereby reducing human errors related to misidentification, ANN were developed to identify Cryptosporidium oocyst and Giardia cyst images. Digitized images of C. parvum oocysts and G. lamblia cysts stained with various commercial IFA reagents were used as positive controls. The images were captured using a color digital camera at 400 x (total magnification), processed, and converted into a binary numerical array. A variety of "negative" images were also captured and processed. The ANN were developed using these images and a rigorous training and testing protocol. The Cryptosporidium oocyst ANN were trained with 1,586 images, while Giardia cyst ANN were trained with 2,431 images. After training, the best-performing ANN were selected based on an initial testing performance against 100 images (50 positive and 50 negative images). The networks were validated against previously "unseen" images of 500 Cryptosporidium oocysts (250 positive, 250 negative) and 282 Giardia cysts (232 positive, 50 negative). The selected ANNs correctly identified 91.8 and 99.6% of the Cryptosporidium oocyst and Giardia cyst images, respectively. These results indicate that ANN technology can be an alternate to having trained personnel for detecting these pathogens and can be a boon to underdeveloped regions of the world where there is a chronic shortage of adequately skilled individuals to detect these pathogens.

Animals↗

PubMed2004

[Detection and identification of the pathogenic cause of a brain abscess by molecular genetic methods].

Brain abscesses are life-threatening and detection and identification of the causative pathogens are crucial for substantiating the diagnosis and for selecting the optimal antibiotic regimen. In approximately 20% of the patients microbiological cultures of abscess material remain sterile. The polymerase chain reaction (PCR) provides a methodological alternative, but data about the use of broad spectrum PCR assays to detect the causative pathogens in brain abscesses are rare. We report on the case of a 65-years-old patient with a brain abscess caused by Fusobacterium spp., which was only diagnosed by broad spectrum PCR. To our knowledge this is the second report about a brain abscess, where Fusobacterium spp. was identified only by broad spectrum PCR and subsequent DNA sequencing.

Aged↗

PubMedDate not supplied

Evaluation of the pediatric Septi-Chek blood culture system.

The pediatric Septi-Chek blood culture system is a biphasic system that uses a 20-ml bottle of brain-heart infusion broth. We compared this system with a supplemented peptone broth tube (Vacutainer) for the recovery of aerobic organisms from blood cultures obtained from patients in a pediatric hospital. Blood specimens for culture were drawn into transport tubes containing sodium polyanetholsulfonate (SPS), and equal volumes were allocated into a Septi-Chek bottle and a Vacutainer tube which was vented for the first day of incubation. A total of 4828 blood culture sets was included, from which 243 probable pathogens were recovered, including 211 in the Septi-Chek system and 204 from the Vacutainer Tube. There were no significant differences in the recovery of individual pathogens. The mean time to initial detection of pathogens was comparable in the two systems, but the mean time to growth on solid media for pathogens recovered in both systems was shorter with Septi-Chek (37.2 hr compared with 45.5 hr, p less than 0.001). The pediatric Septi-Chek system is comparable with a vented Vacutainer tube for the recovery of aerobic pathogens, and its use facilitates the early identification and susceptibility testing of bloodstream pathogens.

Bacteremia↗

PubMed2006

PEMC sensor's mass change sensitivity is 20 pg/Hz under liquid immersion.

To enhance the mass change sensitivity of the resonating piezoelectric-excited millimeter-sized cantilever (PEMC) sensors, we reduced its length and eliminated one layer of its composite structure. As a result the mass sensitivity of the second flexural mode increased by two orders of magnitude (from 10(-9) to 10(-11)g/Hz) and the resonant frequency increased by more than 5 kHz. We demonstrate the effects of modification by detecting a model pathogen Group A Streptococcus (GAS) at 700 cells/mL. The resonant frequency change of the second mode at concentrations of 700, 7 x 10(3), 7 x 10(5), 7 x 10(6), 7 x 10(7), and 7 x 10(9)cells/mL resulted in, respectively, 3.1+/-0.5, 11.6+/-1, 15.7+/-1, 25.7+/-0.15, 28.5+/-2, and 40.5+/-3 ng (n=3 for all) of pathogen attachment. A kinetic model for the binding is proposed and verified. The observed binding rate constant was found to be in the range of 0.051-0.166 min(-1). The significance of the results we report is that the modified PEMC sensors have high mass sensitivity that pathogens can be detected at very low concentration under liquid immersion conditions.

Air↗

PubMed2006

Comparison of four methods of extracting DNA from D. gallinae (Acari: Dermanyssidae).

Dermanyssus gallinae is one of the most serious ectoparasites of poultry and it has been implicated as a vector of several major pathogenic diseases. Molecular detection of such pathogens in mites is crucial and therefore, an important step is the extraction of their DNA from mites. So, we compared four DNA extraction protocols from engorged and unfed individual mites: a conventional method using a Cethyl Trimethyl Ammonium Bromide buffer (CTAB), a Chelex resin, a Qiamp DNA extraction kit and a more recent one filter-based technology (FTA). The DNA samples have been tested for their ability to be amplified by an amplification of a D. gallinae 16S rRNA gene region. The best results were obtained using CTAB and Qiagen methods at the same time with unfed and engorged mites (96% and 100% of amplified samples). FTA produced similar results when using unfed mites but not when processing engorged ones (96% and 70%). Finally, the Chelex method was the least efficient in terms of DNA amplification, especially when applied on engorged individuals (50%). The possible inhibitor role of these Chelex extracted DNA was demonstrated by the means of a PCR control on PUC plasmid. No difference was observed with CTAB, Qiamp DNA extraction kit or FTA methods using DNA extracted one year before.

Animals↗

PubMed2026

Library strategies differentially shape microbial, functional, and host signals in clinical metagenomic sequencing.

Metagenomic next-generation sequencing (mNGS) is increasingly used in infectious disease diagnostics, yet how library preparation shapes the microbial, functional, and host signals recovered from clinical samples remains poorly defined. Here, we performed a within-sample parallel comparison of three mNGS library preparation strategies-DNA-based libraries (DNAlib), RNA-based libraries (RNAlib), and total nucleic acid-based libraries (TNAlib)-across a diverse range of clinical specimens spanning five sample types. Using a curated clinical infectome as a benchmark, we show that library strategies are not interchangeable but capture distinct biological dimensions of the same specimen. RNAlib provided the most comprehensive standalone recovery of the clinical infectome, with improved detection of RNA viruses and cellular pathogens, enhanced resolution of resistance and virulence signals, and preservation of infection-associated host immune signatures. DNAlib showed stronger baseline recovery of DNA viruses and broader host genome coverage, whereas the TNAlib workflow evaluated here largely behaved as an intermediate strategy rather than a consistent improvement over dedicated DNA- or RNA-based workflows. Together, these results establish that the library preparation protocol is a major determinant of how clinical mNGS data should be interpreted and provide a framework for selecting sequencing strategies according to specific diagnostic and biological questions.IMPORTANCEMetagenomic sequencing is increasingly used in infectious disease research and clinical diagnostics, but different library preparation strategies may recover fundamentally different biological signals from the same sample. These signals include not only pathogens but also background microbes, microbial functional activity, and host immune-response patterns. Here, we systematically compared DNA-, RNA-, and total nucleic acid-based metagenomic sequencing libraries using the same clinical samples processed in parallel. We found that the three strategies did not provide equivalent information. RNA-based sequencing generated the most informative single-library view of infection, particularly for RNA viruses, cellular pathogens, functional microbial signals, and host immune-response patterns. DNA-based sequencing was more effective for DNA virus and host genome recovery, whereas the total nucleic acid sequencing workflow evaluated here generally behaved as an intermediate strategy. These findings show that library preparation can substantially influence the interpretation of metagenomic data.

functional characterization↗

PubMed1990

Bronchoscopy specimens in adults with AIDS. Comparative yields of cytology, histology and culture for diagnosis of infectious agents.

Bronchoscopy specimens from 183 known/suspected acquired immunodeficiency syndrome patients were evaluated for pathogens. In each case, transbronchial biopsies were evaluated and bronchoalveolar lavage material was cultured for viruses, fungi and mycobacteria and examined cytologically. A specimen was considered positive for a pathogen if detected by any one of the methods (TBB or BALC or culture). BALC was more sensitive for Pneumocystis carinii than TBB (90 of 92 vs 67 of 80 cases). TBB and BALC had poor sensitivities for cytomegalovirus detection (six of 79 and ten of 91 cases, respectively): 80 of 91 CMV cases were detected by culture only. Nineteen of 26 MB cases were positive by culture only: BALC and TBB detected only three of 26 and five of 23 cases, respectively. Three cryptococcosis cases were detected by culture only. One coccidioidomycosis case was positive by BALC and culture. Culture and BALC in combination detected 212 of 216 all significant pathogens. We believe that TBB is not routinely necessary in AIDS-related bronchoscopies in the absence of suspicion of neoplasia.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction.

OBJECTIVE: To determine the frequencies of human rhinovirus (HRV), respiratory syncytial virus (RSV), and coronavirus (HCV) infection in children with acute otitis media (AOM). METHODS: Middle ear fluids (MEF) collected by tympanocentesis and nasopharyngeal aspirates (NPA) at the time of the AOM diagnosis were examined by reverse transcriptase polymerase chain reaction for HRV, RSV, and HCV RNA. PATIENTS: Ninety-two children aged 3 months to 7 years during a 1-year period. RESULTS: Virus RNA was detected in a total of 69 children (75%) and in 44 MEF samples (48%) and 57 NPA samples (62%) at the time of AOM diagnosis. HRV RNA was detected in both MEF and NPA in 18 (20%), in MEF alone in 4 (4%), and in NPA alone in 10 (11%). RSV was detected in both MEF and NPA in 12 (13%), in MEF alone in 5 (5%), and in NPA alone in 9 (10%). HCV RNA was detected in both MEF and NPA in 5 (5%), in MEF alone in 2 (2%), and in NPA alone in 9 (10%). Dual viral infections were detected in 5% of children. HRV and RSV were detected simultaneously in 2 MEF samples and in 2 NPA samples; RSV and HCV were detected in 1 NPA sample. Bacterial pathogens were detected in 56 (62%) MEF from 91 children. Viral RNA was detected in 20 (57%) MEF of 35 bacteria-negative and in 25 (45%) of 56 bacteria-positive MEF samples. No important differences in the risk of treatment failure, relapse, or occurrence of late secretory otitis media were noted between children with virus-positive and virus-negative MEF aspirates. CONCLUSION: These findings highlight the importance of common respiratory viruses, particularly HRV and RSV, in predisposing to and causing AOM in young children.

Acute Disease↗

PubMed2005

Development of multiplex PCRs for detection of common viral pathogens and agents of congenital infections.

Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogen-positive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10(2) copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.

Amniotic Fluid↗

PubMed2005

Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR.

BACKGROUND: Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed. RESULTS: We designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background. CONCLUSION: The methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities.

Ascomycota↗