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PubMed1997

Prevalence and characteristics of severe rotavirus infections in Nicaraguan children.

We analyzed the prevalence of rotavirus in 296 children age between 3 and 36 months who were hospitalized in 1994 with severe gastro-enteritis at two health centres for diarrhoea treatment in León, Nicaragua. Enteric viruses were detected in 96 (32.4%) of the children and rotaviruses were the most common pathogens detected in 84 (28%). The majority of rotavirus infections occurred in children less than 1 year old and all strains isolated belonged to subgroup II and had 'long' RNA patterns. Molecular epidemiology of 55 rotavirus strains revealed that all had the same RNA migration pattern and serotyping of 37 strains by PCR technology revealed that all isolates belonged to serotype 3. A significant observation was that only one electropherotype of rotavirus circulated. No non-group A rotaviruses were found by RNA gel electrophoresis. Adenoviruses were found by ELISA in 14 of 265 (5%) children and were most frequently detected during the 1st year of life. Of 103 faecal samples analyzed by electron microscopy, four contained small round structured viruses.

Adenovirus Infections, Human↗

PubMed1994

Identification of pathogenic leptospires by recombinant DNA probes.

Early diagnosis of leptospirosis of pulmonary diffuse hemorrhage type (PDH) is of crucial importance in saving patients. To develop a sensitive and specific method for diagnosis, a genomic library of the main pathogen of PDH, L. interrogans serovar lai strain 017, was constructed with the plasmid vector pUC9. Recombinant plasmids which have homologous fragments of pathogenic leptospires were screened from the bank. A recombinant plasmid, designated pCX7, could detect 1.7 kb fragment of strain 017, 9.0 kb of strain 601 and 30.0 kb of strain Hebdomadis, respectively, without cross hybridization with nonpathogenic leptospires such as L. biflexa strain Patoc I and Leptonema illini. The recombinant plasmid pCX7 could detect pathogenic leptospires which are the main pathogens endemic to Sichuan Province.

Blotting, Southern↗

PubMed1996

[The etiology of acute lower respiratory tract infections in infants].

The etiology of acute lower respiratory tract infections (ALRI) was studied in pediatric inpatients under 2 years of age admitted to Chiba Municipal Hospital between June 1994 and March 1995. Eighty-seven patients, 99 episodes were investigated for bacterial infection with the use of blood culture and washed sputum culture, for viral infection with the use of virus isolation, antigen detection and antibody assays, for Mycoplasma pneumoniae infection with the use of antibody assay and for Chlamydia infection with the use of antigen detection. Pathogens were identified in 71 (71%) of the 99 episodes. Evidence of bacterial infection was detected in 43 episodes (43%), viral infection in 37 episodes (37%), Mycoplasma pneumoniae infection in 4 episodes (4%) and Chlamydia infection 3 episodes (3%). The major bacterial pathogens were H. influenzae, M. (B) catarrhalis and S. pneumoniae. RS virus and influenza virus epidemics occurred during the winter. A mixed bacterial and viral infection was documented in 13 episodes (13%). RS virus infection was common in infants up to 6 months old. Mixed bacterial and influenza virus infections were common in 1 or more year old children. Virus isolation was useful for the grasp of the viral epidemic. Bacterial associated infections were common in children under 2 years of age with ALRI. Washed sputum culture and sputum gram stains' were useful for the treatment of infant ALRI.

Acute Disease↗

PubMed1992

Aetiological studies on hospital inpatients with secretory diarrhoea in Calcutta.

Twenty-five cases of all age groups with secretory diarrhoea admitted to the Infectious Diseases Hospital, Calcutta were studied. Bacterial enteropathogens were detected by bacteriological examination of the faeces, in each and every case. The main pathogens detected were Escherichia coli and Vibrio cholerae. V cholerae was more commonly associated with severe illness except in the very young, while Esch coli was equally associated with moderate and severe illness. Multiple isolates were more commonly associated with severe illness and single isolates were more common in illness of moderate severity.

Adolescent↗

PubMed1991

Etiology of childhood diarrhea in Beijing, China.

To determine the role of recently recognized enteropathogens in childhood diarrhea in China, 221 children with diarrhea and 108 controls seen at the Beijing Children's Hospital were studied during April and May 1989. Stools were examined for ova, parasites, and rotavirus, cultured for bacterial pathogens, and probed for enterotoxigenic Escherichia coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and enteropathogenic adherence factor-positive (EAF+) E. coli. Pathogens were identified in 56.5% of children with diarrhea and 43.5% of controls (P = 0.04). Detection of enteropathogens was significantly greater in patients examined within 1 week of symptom onset (65%) than in patients examined later (39%; P = 0.01). ETEC was the most frequently detected pathogen in children with diarrhea, accounting for 20% of the cases. Other agents identified in patients included the following: salmonellae, 12%; rotavirus, 7%; EIEC, 7%; EHEC, 7%; members of the Aeromonas hydrophila group, 6%; EAF+ E. coli, 5%; Ascaris lumbricoides, 3%; shigellae, 3%; campylobacters, 2%; and Vibrio spp., 0.5%. The isolation rates of salmonellae (P = 0.02), EAF+ E. coli (P = 0.04), and mixed pathogens (P = 0.05) were significantly greater for diarrhea patients than for controls. Resistance to multiple antimicrobial agents occurred in 39% of the Salmonella isolates, 22% of the Aeromonas isolates, and 17% of the Shigella isolates. Multiresistant salmonellae (P = 0.05) and shigellae were recovered from diarrheal stools only. Ciprofloxacin, cefotaxime, and imipenem were the only agents tested to which all bacterial isolates were susceptible in vitro. These results suggest that both traditional and newly recognized agents are important causes of childhood diarrhea in Beijing and that therapy may be complicated by indigenous antimicrobial resistance.

Child, Preschool↗

PubMed1997

[Molecular pathologic detection of mycobacteria].

In recent years, significant advances in the diagnosis of mycobacterial infections have been made by the introduction of direct pathogen detection methods. These techniques are usually based on the polymerase chain reaction (PCR) or on a transcription-mediated amplification (TMA) process. The majority of the protocols have been optimized for the detection of mycobacterial nucleic acids in fresh fluid or fresh tissue specimen. Unfortunately pathologists are frequently confronted with the problem that tissues with histologically suspicious lesions have been entirely fixed in formalin. As a result of this routine fixation, DNA and RNA are heavily degraded and the usually high sensitivity of the amplification techniques is greatly impaired. Consequently, only PCR protocols designed for small amplification targets are still suitable for an efficient detection of microbial DNA in formalin-fixed and paraffin-embedded tissues. We therefore adapted PCR assays with amplification products < 200 bp for the detection of M. tuberculosis-complex DNA (targets: IS6110 and 65 kDa-antigen gene) in routine biopsies. Although the sensitivities of the two assays varied significantly with the degree of DNA degradation, we were able to detect M. tuberculosis-complex specific DNA in about 25% of the tissues with a granulomatous inflammation and negative Ziehl-Neelson stain. Recently, we have added a third PCR-assay, which in combination with direct sequencing also allows us to detect DNA from M. leprae and several atypical mycobacteria species. PCR-analysis has significantly improved the diagnosis of mycobacterial infections by supplementing conventional histological examination of formalin-fixed and paraffin-embedded tissues.

DNA, Bacterial↗

PubMed1993

Diarrhoeal disease: current concepts and future challenges. Molecular biological approaches to the epidemiology of diarrhoeal diseases in developing countries.

Diarrhoea in developing countries is caused by an increasingly long list of bacterial, viral, and parasitic pathogens with rotavirus, enterotoxigenic Escherichia coli, Campylobacter, Shigella, and Salmonella heading the list. Using methods to detect most of the known enteropathogens, one or more enteropathogen(s) is isolated in two-thirds of diarrhoeal illnesses in the developing world. Many of these enteropathogens are also frequently isolated from children without diarrhoea. An aetiologic agent is more frequently isolated from cases of invasive diarrhoea than from those with secretory diarrhoea. Deoxyribonucleic acid probes have proved very useful in detecting pathogens such as enterotoxigenic (ETEC), enteroinvasive (EIEC), and enteropathogenic E. coli (EPEC), but have not yet proved to be particularly rapid or less expensive. Molecular biology has proved useful in epidemiological studies as a means of strain identification. Plasmids were initially used as convenient markers and proved useful in identifying epidemic strains of bacteria. Other molecular markers, such as ribotyping, are accurate enough to be used as taxonomic tools.

Bacterial Infections↗

PubMed1978

Isolation of Treponema hyodysenteriae from sources other than swine.

Fecal samples were collected from animals and environments on 3 swine farms and cultured for Treponema hyodysenteriae. Each farm was a farrow-to-finish operation and, at the time of sampling, swine dysentery was enzootic among 8- to 22-week-old pigs. Pathogenic T hyodysenteriae was isolated from pigs on all 3 farms. On farm A, nonpathogenic T hyodysenteriae was isolated from a sample of lagoon water. On farm B, pathogenic T hyodysenteriae was isolated from a waste-holding pit. On farm C, a dog was observed to be eating feces of pigs that had swine dysentery. The dog was diarrheic and a fecal sample yielded a pathogenic isolant of T hyodysenteriae. Further isolation attempts were unsuccessful after the dog was removed from the infected premises. Isolation of pathogenic and nonpathogenic organisms from waste-holding systems emphasizes the need for cultural techniques in detecting pathogenic T hyodysenteriae.

Animals↗

PubMed1997

Molecular diagnosis of Ureaplasma urealyticum in an immunocompetent patient with destructive reactive polyarthritis.

Polymerase chain reaction (PCR) amplification, which is a useful method for detecting infectious agents in joints, has potential utility in the molecular diagnosis of venereal-associated arthritis. Among pathogens detected by this technique, Ureaplasma urealyticum, which is primarily associated with reactive arthritis (ReA), is also implicated in septic arthritis in immunocompromised patients. We report here a case of destructive polyarthritis, initially suggestive of septic arthritis, in an immunocompetent patient whose PCR positivity for U. urealyticum DNA in one joint, in conjunction with the disease outcome and histologic findings, led to the diagnosis of destructive ReA.

Adult↗

PubMed1987

Clinical relevance of culture versus screens for the detection of microbial pathogens in urine specimens.

The use of screens to detect "significant levels" of pathogenic microorganisms in urine specimens offers the advantages of both rapidly reporting results and controlling costs. Many of these screens, however, are insensitive at microbial counts below 10(5) colony-forming units (CFU)/ml of urine. It is increasingly apparent that patients with almost any type of urinary tract infection (except for most patients who are asymptomatic or who have pyelonephritis) may have urine concentrations of pathogens as low as 10(2) to 10(3) CFU/ml. This review documents factors that can contribute to diminished concentrations of microorganisms in urine, lists patient populations in whose urine microorganisms in concentrations well below 10(5) CFU/ml have been associated with infection, and makes recommendations for selection of laboratory tests, including rapid screens, for the diagnosis and management of urinary tract infections.

Age Factors↗

PubMedDate not supplied

[Culture media for the detection and the identification of Streptococcus agalactiae].

Streptococcus agalactiae, a Group B streptococcus, is the main cause of bacterial perinatal infection and is also an important opportunistic pathogen. Detection and identification of S. agalactiae are straight forward with special culture media, where Group B streptococci show a specific, typical pink or red pigment. To quickly and easily detect the pigment, culture media should contain: (i) starch; (ii) an inhibitor of the folate pathway; (iii) animal serum; (iv) a pepsic proteic hydrolysate; and (v) glucose, together with a high-capacity buffer. When selective antibiotics are added to culture media designed in this way, it is possible to detect S. agalactiae directly from clinical samples by observation of its pigment after less than 12 hours of aerobic incubation.

Culture Media↗

PubMed1994

Leukocyte esterase urine strips for the screening of men with urethritis--use in developing countries.

BACKGROUND AND OBJECTIVES: The leukocyte esterase (LE) strip is a useful tool for the screening of men with urethritis. In developing countries, where laboratory facilities are limited, and sexually transmitted diseases endemic, simple and inexpensive diagnostic tests which perform well, would be of great value. METHODS: Men presenting with urethritis to a referral clinic for sexually transmitted diseases in Nairobi, Kenya participated in this cohort analytical study. First-void urine was collected for LE dipstick testing as part of the diagnostic work-up. The results of the dipstick measurement were compared with the laboratory detection of Chlamydia trachomatis and Neisseria gonorrhoeae. RESULTS: Of 200 men with symptoms of urethritis, 33 (17%) had a pathogen detected from the urethra or the urine. Chlamydia was detected in urine by PCR in 22 (11%), and gonorrhoea was cultured from the urethra in 11 (6%). Esterase activity (trace or greater) had a sensitivity of 76%, a specificity of 80%, a positive predictive value of 42% and a negative predictive value of 94% for the presence of chlamydia or gonorrhoea. CONCLUSIONS: The use of the LE dipstick for the screening of men with symptomatic urethritis can improve diagnostic accuracy and reduce the amount of empiric antimicrobial therapy. The low detection rate of chlamydia in these men with a clinical diagnosis of nongonococcal urethritis needs further study.

Adult↗

PubMed2026

Using Mapping-Profiles to Refine Strain-Level Metagenomic Classification.

Metagenomic classification at the strain level remains challenging due to high sequence similarity among closely related genomes, which leads to ambiguous read mappings and frequent false-positive strain detections. Reducing such errors improves the reliability of strain-level analyses, which is critical for applications such as pathogen detection. We introduce StrainRefine, a post-mapping refinement method that analyzes read-reference mapping profiles to resolve ambiguous assignments among highly similar genomes. The method represents candidate reference genomes using binary profiles that capture read-support patterns and measures similarity between references based on profile overlap. The method clusters references based on similar mapping profiles, filters weakly supported genomes, and reassigns reads to representative references, reducing redundant reporting of near-identical strains. StrainRefine substantially reduces false-positive strain detections while preserving recall and improving agreement between predicted and true abundance profiles. On large-scale metagenomic datasets, it achieves a substantially improved precision-recall balance compared with existing mapping-based approaches, with the standalone method obtaining the highest read-level classification accuracy on the most complex evaluated dataset. Unlike many strain-level tools designed for individual species, StrainRefine operates without prior assumptions about sample composition or curated species-specific reference collections, while still achieving comparable performance in single-species settings on species-specific reference databases. These results highlight mapping-profile similarity as an effective signal for improving strain-level metagenomic classification.

false-positive reduction↗

PubMed1998

Endoscopic evaluation of chronic human immunodeficiency virus-related diarrhea: is colonoscopy superior to flexible sigmoidoscopy?

OBJECTIVES: In patients with chronic human immunodeficiency virus (HIV)-related diarrhea undergoing lower endoscopy, the decision to perform flexible sigmoidoscopy or colonoscopy is controversial. The purpose of this study is twofold: 1) to evaluate the diagnostic yield of colonoscopy in a large group of patients with chronic HIV-related diarrhea and negative stool studies, and 2) to determine whether colonoscopy is superior to flexible sigmoidoscopy in this setting. METHODS: All HIV-infected patients with chronic diarrhea who were referred for diagnostic colonoscopy at Bellevue Hospital Center between January 1992 and December 1996 were identified. Patient charts, pathology reports, and endoscopy records were reviewed. RESULTS: During the 5-yr study period, 317 consecutive patients with chronic unexplained diarrhea undergoing colonoscopy were identified. A potential cause of diarrhea was found in 116 patients (36.6%). Cytomegalovirus was the most common pathogen detected (24%). The yield of colonoscopy was significantly higher in patients with a CD4 count of <100 cells/mm3 than in those with higher CD4 counts (44.8% vs 6.4%, p < 0.0001). Thirty percent of pathogens and 75% of lymphomas were identified only on biopsies taken from the proximal colon, well beyond the reach of the flexible sigmoidoscope. Importantly, 94% of the pathogens that were found only in the proximal colon were organisms for which effective therapy is currently available. CONCLUSIONS: Colonoscopy is superior to flexible sigmoidoscopy in HIV-infected patients with chronic unexplained diarrhea. If flexible sigmoidoscopy had been performed instead of colonoscopy, 30% of pathogens would have been missed and 75% of lymphomas would have escaped detection.

AIDS-Related Opportunistic Infections↗

PubMed1992

A DNA probe specific to pathogenic Entamoeba histolytica.

A DNA sequence, IE-gen1 (3.1 kb), was isolated from the pathogenic strain of E. histolytica NIH-200. IE-gen1 was identified by the subtractive hybridization of a genomic library to a cDNA probe prepared from NIH-200 trophozoites. The IE-gen1 probe specifically detected pathogenic E. histolytica in slot blots of genomic DNA and Northern blots, but not other Entamoeba species and additional human parasites. This genomic probe could detect with complete specificity DNA from about 10(3) organisms. The IE-gen1 probe could be related to highly specialized loci in pathogenic E. histolytica, and is likely to be a valuable DNA reagent for clinical diagnosis and epidemiological investigations.

Animals↗

PubMed1996

Enzyme-linked immunomagnetic electrochemical detection of Salmonella typhimurium.

There is a need for rapid methods to detect pathogenic bacteria in food products as alternatives to the current laborious and time-consuming culture procedures. We report a microbial detection technique that combines the selectivity of antibody-coated superparamagnetic beads with the rapidity and sensitivity of electrochemical detection in a format termed enzyme-linked immunomagnetic electrochemistry. In it, Salmonella typhimurium were sandwiched between antibody-coated magnetic beads and an enzyme-conjugated antibody. With the aid of a magnet, the beads (with or without bound bacteria) were localized onto the surface of disposable graphite ink electrodes in a multi-well plate format. Enzyme substrate was added and conversion of substrate to an electroactive product was measured using electrochemical detection. The electrochemical response was directly proportional to the number of captured bacteria. Using this technique, a minimum detectable level of 8 x 10(3) cells/ml of Salmonella typhimurium in buffer was achieved in ca. 80 min.

Electrochemistry↗

PubMed1995

A rapid method to determine bacterial contamination on hatching eggs. 3. Use of commercial DNA probe kits for detection of specific pathogens after six hours of incubation.

The usefulness of commercially available DNA probe kits for the detection of Escherichia coli and Salmonella spp. after only 6 h of incubation, was determined. It was established that the commercially available probe kits used could detect E. coli at initial levels of approximately 4.5 x 10(2) colony-forming units (cfu) per ml after only 6 h of incubation in nutrient broth (NB). Initial bacterial levels as low as 4.5 x 10(1) cfu/ml could be detected when the NB was incubated for 18 h. Salmonella Enteritidis, at initial levels of 2.86 x 10(2) cfu/ml could be detected after 6 h of incubation at 37 degrees C in NB, while initial levels as low as 2.86 x 10(-1) cfu/ml could be detected after 18 h at 37 degrees C in both NB and selected media, as specified by the manufacturers of the probe kits. Commercially available DNA probe kits can therefore be used to detect specific pathogens on the surface of hatching eggs and these probes can be used in conjunction with an egg-washing system, which is used to determine total bacterial contamination, although a longer incubation period greatly improves the sensitivity of these tests.

Animals↗

PubMed1997

Impact of molecular biology on the detection of foodborne pathogens.

Molecular biological methods that use antibodies and nucleic acids to detect specific foodborne bacterial pathogens were scarcely known a decade and a half ago. Few scientists could have predicted that these tools of basic research would come to dominate the field of food diagnostics. Today, a large number of cleverly designed assay formats using these technologies are available commercially for the detection in foods of practically all major established pathogens and toxins, as well as of many emerging pathogens. These tests range from very simple antibody-bound latex agglutination assays to very sophisticated DNA amplification methods. Although molecular biological assays are more specific, sensitive, and faster than conventional (often cultural) microbiological methods, the complexities of food matrices continue to offer unique challenges that may preclude the direct application of these molecular biological methods. Consequently, a short cultural enrichment period is still required for food samples prior to analysis with these assays. The greater detection sensitivity of molecular biological methods may also affect existing microbiological specifications for foods; this undoubtedly will have repercussions on the regulatory agencies, food manufacturers, and also consumers.

Antibodies, Bacterial↗