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PubMed2005

Isolation of Vibrio cholerae from neonates admitted to an urban diarrhoeal diseases hospital in Bangladesh.

BACKGROUND: Very little is known about Vibrio cholerae infection in neonates with diarrhoea. AIM: The aim was to examine the frequency of enteropathogenic bacteria, especially Vibrio cholerae, in stool specimens from neonates with diarrhoea. METHODS: We reviewed the hospital records of 240 neonates admitted with diarrhoea to the Dhaka Hospital of ICDDR,B: Centre for Health and Population Research, Dhaka, Bangladesh during January-December 2001. On admission, rectal swabs or stool specimens were plated directly onto taurocholate-tellurite-gelatin agar, Salmonella-Shigella agar and MacConkey's agar for culture of Vibrio cholerae, Shigella and Salmonella spp. RESULTS: A single bacterial enteric pathogen was detected in 71 neonates, multiple pathogens in 12, and stool samples of 157 neonates revealed no pathogen. The identified enteropathogens were as follows: V. cholerae in 42 neonates, Shigella spp in 22, Salmonella spp in 8, Aeromonas spp in 9 and Hafnia alvei in 2. CONCLUSION: V. cholerae is not uncommon in neonates presenting with diarrhoea in cholera-endemic countries.

Aeromonas↗

PubMed2002

DNA probes and primers in dental practice.

In clinical microbiology, molecular genetic techniques are increasingly being used to detect and/or differentiate uncultivable, anaerobic, or fastidious microorganisms. During the past decade, DNA probe hybridization and in vitro amplification by polymerase chain reaction have also been introduced to detect oral pathogens. The present review describes experiences with methods and commercial test systems for the detection of pathogens in periodontitis and caries.

Bacteria↗

PubMed2005

Universal primers for detection of common bacterial pathogens causing prosthetic joint infection.

The diagnosis of low grade prosthetic joint infection is difficult and time consuming. Nested-PCR for universal bacterial DNA segments detection of "orthopaedic" bacteria was tested in a laboratory setting. This method is based on amplification of the 16S bacterial ribosomal RNA coding sequences. 11 species of the most frequent bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens) involved in prosthetic joint infections were studied. All could be detected rapidly and sensitively by this method.

Bacteria↗

PubMed1992

Serodiagnosis of bacterial diseases: problems and developments.

Serodiagnosis is useful when pathogens cannot be detected quickly or easily by conventional methods or have been eliminated by the patient. Taking the example of leptospirosis, the microscopic agglutination test to detect specific antibodies after leptospires have disappeared from the patient's bloodstream is of unsurpassed reliability. Unfortunately the test is used mainly in specialized laboratories that can culture leptospires to serve as live antigens. Freeze-dried leptospires may allow the application of the test on a wide scale. The ELISA is useful for the detection of IgM antibodies in humans as a sign of current or recent infection. ELISA results must be confirmed by the microscopic agglutination test. A simplified easy-to-read ELISA using broadly reactive antigen for the detection of all types of leptospires might be useful for a quick detection of acute human leptospirosis. Using an appropriate antigenic preparation the ELISA is useful for the detection of antibodies in animals, e.g. cattle. As for pathogen detection, DNA-based methods such as PCR are promising for an early, quick and specific diagnosis. Antigenic analysis by monoclonal antibodies and genetic analysis by restriction fragment length polymorphism are for the time being complementary methods for the characterization of leptospiral isolates.

Antibodies, Bacterial↗

PubMed2003

Use of polymerase chain reaction to detect the soft rot pathogen, Pythium myriotylum, in infected ginger rhizomes.

AIMS: The aims are to establish a polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in the rhizome of ginger and diagnosing ginger soft rot and screening health seed ginger. METHODS AND RESULTS: A booster PCR method was established for detection of P. myriotylum using a specific primer selected from rDNA ITS1 region coupled with universal primer ITS2. It successfully applied to the detection of P. myriotylum in naturally infected ginger rhizomes but not from DNA of ginger rhizomes collected from field without target fungus. CONCLUSIONS: A specific method for detecting P. myriotylum was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The new PCR method has allowed us to monitor ginger for the presence of P. myriotylum as a way of disease diagnosis or healthy seed ginger examination.

Zingiber officinale↗

PubMed1996

Empirical treatment of severe acute community-acquired gastroenteritis with ciprofloxacin.

We conducted a randomized controlled trial to determine whether empirical treatment of severe acute community-acquired gastroenteritis (four fluid stools per day for > 3 days) with ciprofloxacin reduces the duration of diarrhea and other symptoms and to determine what effect ciprofloxacin has on the duration of long-term fecal carriage of gastrointestinal pathogens. A total of 173 patients were recruited for the study and received either ciprofloxacin (500 mg b.i.d.) or placebo for 5 days, during which time they recorded the duration of diarrhea and other symptoms (fever, abdominal pain, vomiting, and myalgia). Fecal samples were collected before treatment and regularly after treatment to determine the duration of carriage of gastrointestinal pathogens. Antibiotic susceptibility tests were performed, and the minimum inhibitory concentrations (MICs) of ciprofloxacin were determined. A significant reduction in the duration of diarrhea and other symptoms was observed after treatment, regardless of whether a pathogen was detected (P = .0001). Treatment failure occurred in 3 of 81 patients in the ciprofloxacin group and 17 of 81 patients in the placebo group. Significant pathogens were detected in 87% of patients, 85.5% of whom had cleared the pathogen at the end of treatment with ciprofloxacin, as compared with 34% who received placebo. Six weeks after treatment, there was no difference between the two groups in terms of the pathogen carriage rate (12%). Treatment with ciprofloxacin did not prolong carriage. High-level resistance to ciprofloxacin (MIC, > 32 mg/L) was detected in three strains (4%) of Campylobacter species.

Adult↗

PubMed2004

Multipathogen oligonucleotide microarray for environmental and biodefense applications.

Food-borne pathogens are a major health problem. The large and diverse number of microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. Some of these pathogens and their toxins have potential use as bioweapons. DNA microarray technology allows the simultaneous analysis of thousands of sequences of DNA in a relatively short time, making it appropriate for biodefense and for public health uses. This paper describes methods for using DNA microarrays to detect and analyze microbial pathogens. The FDA-1 microarray was developed for the simultaneous detection of several food-borne pathogens and their virulence factors including Listeria spp., Campylobacter spp., Staphylococcus aureus enterotoxin genes and Clostridium perfringens toxin genes. Three elements were incorporated to increase confidence in the microarray detection system: redundancy of genes, redundancy of oligonucleotide probes (oligoprobes) for a specific gene, and quality control oligoprobes to monitor array spotting and target DNA hybridization. These elements enhance the reliability of detection and reduce the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray. The results presented demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefense relevant microbial pathogens.

Bacteria↗

PubMed2003

Methods for rapid separation and concentration of bacteria in food that bypass time-consuming cultural enrichment.

The rapid detection of pathogenic organisms that cause foodborne illnesses is needed to insure food safety. Conventional methods for the detection of pathogens in foods are time-consuming and labor-intensive. New advanced rapid methods (i.e., polymerase chain reaction, DNA probes) are more sensitive and selective than conventional techniques, but many of these tests are inhibited by food components, rendering them dependent on slow cultural enrichment. The need for alternative methods that will rapidly separate and concentrate bacteria directly from food samples, thereby reducing the time required for these new rapid detection techniques, is evident. Separation and concentration methods extract target bacteria from interfering food components and/or concentrate bacteria to detectable levels. This review describes several methods used to separate and/or concentrate bacteria in food samples. Several methods discussed here, including centrifugation and immunomagnetic separation, have been successfully used, individually and in combination, to rapidly separate and/or concentrate bacteria from food samples in less time than is required for cultural enrichment.

Bacteria↗

PubMed2001

Detection of putative periodontal pathogens in non-insulin-dependent diabetes mellitus and non-diabetes mellitus by polymerase chain reaction.

It has been assumed that there is a relationship between periodontal diseases and diabetes mellitus, however the putative periodontal microorganisms in non-diabetes mellitus (non-DM) individuals and non-insulin-dependent diabetes mellitus (NIDDM) patients have not been well studied. In this study, the detection rates of 5 putative periodontal pathogens: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens, Treponema denticola, and Candida albicans by polymerase chain reaction (PCR) between NIDDM and non-DM adults were compared. A total of 246 adults were randomly recruited and periodontal parameters including: plaque index (P1I), gingival index (GI), probing depth (PD) and attachment level (AL) were recorded. Subgingival plaque samples were collected by sterile curettes from the most diseased and healthy sites based on PD and AL. The differences in periodontal parameters and microbiological data in healthy and diseased sites between non-DM and NIDDM patients were compared by chi-square analysis. The results showed no significant differences in age, gender, GI, P1I, PD, and prevalence of the 5 microorganisms between the NIDDM and the non-diabetic groups. However, except for A. actinomycetemcomitans, the prevalence of the periodontal microorganisms tested was significantly higher (p <0.001) in diseased sites than in the healthy sites in both groups. The P1I, GI, PD and AL were significantly higher in T. denticola positive sites than in negative sites. The results suggested that P. gingivalis, T. denticola, E. corrodens and C. albicans may play important roles in the periodontitis of both NIDDM and non-DM individuals, however the etiology of periodontitis in both groups may not be different from each other.

Adult↗

PubMed2004

Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients.

A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.

Adolescent↗

PubMed2006

Systemic movement of Agrobacterium tumefaciens in several plant species.

AIMS: The systemic movement of Agrobacterium spp. inside plants of different species was studied to determine the most valuable diagnostic methodology for their detection. METHODS AND RESULTS: Pathogenic agrobacteria were detected by isolation and PCR in tissue away from primary tumours in tomato plants grown in the presence of Agrobacterium spp. Moreover, this bacterium was also able to induce secondary tumours beyond the inoculation site. In addition, the capacity of agrobacteria to translocate and induce secondary tumours was analysed in rose, grapevine, chrysanthemum, cherry and peach x almond hybrid GF677. No differences among strains of Agrobacterium spp. were detected in secondary tumour development, although some of them induced a significantly higher number of primary tumours in some species. Movement of inoculated pathogenic cells of four strains was also demonstrated in symptomless portions of the plant stems by isolation and PCR. Finally, pathogenic agrobacteria were detected in root, crown and stem portions of naturally infected walnuts. In all assays, PCR was the most efficient technique for detecting the movement of Agrobacterium spp. within the plants. CONCLUSIONS: Migration of agrobacteria inside plants is a complex phenomenon and more extensive than previously reported. Therefore, efficient and sensitive detection methods such as PCR must be used to select clean plants to avoid latent infections of Agrobacterium spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that migration of Agrobacterium spp. could be relatively frequent in several cultivated fruit trees, and systemic infections should be taken into account when designing strategies for controlling crown gall disease.

Agrobacterium tumefaciens↗

PubMed1996

Investigation of chronic diarrhoea in acquired immunodeficiency syndrome. A prospective study of 155 patients.

BACKGROUND AND AIMS: The optimum diagnostic investigation for patients with acquired immunodeficiency syndrome (AIDS) and diarrhoea is not known. Often no pathogen is detected and it is unclear whether this is because pathogens are absent in some patients or the investigations used fail to detect them. The hypothesis that AIDS related diarrhoea is usually due to an infection, which can be identified by a simple diagnostic strategy based on the results of intensive investigation of a cohort of such patients, was investigated. METHODS: 155 patients with AIDS and chronic diarrhoea underwent contemporaneous examination of stools, duodenal, jejunal, and rectal biopsy specimens and duodenal aspirate for bacterial, protozoal, and viral pathogens. A decision tree analysis was used to determine the best sequential diagnostic strategy for clinicians. RESULTS: 128 of 155 patients investigated (83%) had at least one potential pathogen. The presenting clinical features could not predict the presence or site of the pathogens. Stool analysis identified the most pathogens (93 of 199, 47%). Rectal biopsy was essential for the diagnosis of cytomegalovirus and adenovirus. Duodenal biopsy was as helpful as jejunal biopsy and detected some treatable pathogens missed by other methods. Electron microscopy, impression smears, and duodenal aspirate yielded little extra information. If gut biopsy was reserved for patients without a stool pathogen, some treatable pathogens would have been missed. CONCLUSION: Most patients with AIDS and chronic diarrhoea have at least one gut pathogen, which can be identified by stool analysis and light microscopic examination of duodenal and rectal biopsies. Some pathogens will be missed unless all these investigations are done on all such patients.

Acquired Immunodeficiency Syndrome↗

PubMed2002

[Laboratory tests for infectious diseases and medical reimbursement].

Emerging and reemerging infectious diseases, drug-resistant bacteria and nosocomial infection are becoming increasingly serious social problems. To control infectious diseases, it is important to develop effective therapies and to achieve complete prevention of pathogen transmission. For these purposes, rapid detection of pathogenic microorganisms is essential. Recent development and technical innovation in laboratory tests and diagnosis for infectious diseases are remarkable. New techniques such as antigen detection and genetic diagnosis, neither of which require conventional culture, have been intensively developed and introduced into clinical practice. These methods are now widely accepted as effective diagnostic tools because of their high specificity, high sensitivity and rapidity. In this article, we describe the burden of medical expenses under the law on novel infectious diseases and medical reimbursement for diagnostic tests for these diseases.

Clinical Laboratory Techniques↗

PubMed1998

rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7.

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens, Escherichia coli O157:H7 and enterotoxigenic Staphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37 degrees C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1, 400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121 degrees C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80 degrees C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

Blotting, Northern↗

PubMed2005

Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.

Fish and shellfish diseases are a constant threat to the sustainability and economic viability of aquaculture. Early diagnosis plays a vital role in management of fish and shellfish diseases. Traditionally, various biochemical and serological tests have been used for fish disease diagnosis. However, the time and expertise required for such diagnoses makes it difficult for aquaculturists to easily adopt them under production conditions. Polymerase chain reaction and probe-based nucleic acid detection have become increasingly popular in fish and shellfish diagnostics. Recently, a novel technique called loop-mediated isothermal amplification (LAMP) has been developed, which is highly sensitive and rapid. LAMP has been used for the detection of bacterial, viral, fungal and parasitic diseases in both animal and plants. In aquaculture, LAMP-based detection of pathogens like Edwardsiella tarda, E. ictaluri, Nocardia seriolae, Tetracapsuloides bryosalmonae, white spot syndrome virus and infectious haematopoietic necrosis virus have been reported. In this review, the application of LAMP for the detection of aquaculture-associated pathogens is discussed.

Animals↗

PubMed2004

Nosocomial diarrhea.

OBJECTIVE: To study the occurrence of nosocomial diarrhea in pediatric wards and the role of infections in its causation. METHODS: 75 cases conforming to the CDC definition of nosocomial diarrhea were selected for the study. Stool samples were subjected to direct microscopic examination, bacteriological culture and direct antigen detection for Clostridium difficile (C. difficile) and Rota virus. RESULTS: All the cases were sporadic in origin. Rota virus was detected in 16.67% cases and Enteropathogenic Escherichia coli (E.P.E.C.) was detected in 12% cases. Cases in which no pathogen was detected (82.7%) outnumbered those in which a pathogen was identified (17.3%). CONCLUSION: Rota virus and E.P.E.C were the most common etiological agents. C. difficile was detected in one case only. Most of the sporadic cases of nosocomial diarrhea in pediatric patients can be managed on fluids and electrolytes.

Child, Preschool↗

PubMed2004

Correlations between phenotype and microsatellite instability in HNPCC: implications for genetic testing.

Hereditary nonpolyposis colorectal cancer (HNPCC) is widely considered to be a syndrome of defective mismatch repair (MMR). A major concern with genetic diagnosis of HNPCC is the variable, often low, percentage of pathogenic germline mutations that can be detected in MMR genes using common screening methods. The variable percentage of mutation detected is in part related to the sensitivity of conventional screening methods and may also depend on the heterogeneous genetics of HNPCC. Thus, identification of phenotypic criteria predictive of germline mutations in MMR genes may be helpful in efficient HNPCC genetic testing. Clinical diagnostic criteria, initially developed for HNPCC (e.g., Amsterdam I and II, or Bethesda criteria), can be used to clinically select patient candidates that carry germline mutations in MMR genes. More useful criteria were previously developed by analyzing families with germline MMR mutations. Using a complementary approach based on tumor microsatellite instability analysis, we confirm that the Amsterdam criteria are significantly better than the Bethesda criteria in predicting families with MSI-H tumors (P = 0.0227). Our results also suggest that a cutoff at < 50 years' mean age at diagnosis of HNPCC-related cancers (especially colorectal and endometrial cancer) may be an additional tool for the identification of families with defective MMR. Recent advances in MMR mutation screening are expected to improve detection of pathogenic MMR mutations in these families. Conversely, the high proportion of MSS tumors observed in our series of families with advanced age at cancer diagnosis is consistent with the low percentage of MMR mutations detected by previous studies in families with this phenotype. These families probably carry mutations in other genes that may or may not be related to MMR. Additional studies are necessary to clarify the molecular basis for HNPCC in families with MSS tumors.

Adult↗

PubMed2004

Application and evaluation of RT-PCR-ELISA for the nucleoprotein and RT-PCR for detection of low-pathogenic H5 and H7 subtypes of avian influenza virus.

Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT-PCR-ELISA) and 2) the subtype specific RT-PCR for H5 and H7 were compared to the results obtained by inoculation of the same specimens into the allantoic cavity of embryonated specific pathogen free (SPF) hen's eggs. Using inoculation in SPF fowl eggs as standard the sensitivity of the NP RT-PCR-ELISA and the RT-PCR for H5 or H7 was 91% and 94%, and the corresponding specificity 98% and 96%. In comparison with inoculation into eggs an additional of 9 samples were positive by NP RT-PCR-ELISA and 13 samples were positive by RT-PCR for one of the HA subtypes. Hence, the 3 RT-PCR procedures described are fast, sensitive and specific for detecting AIV and subtyping H5 and H7 and they are obvious alternatives when testing large numbers of samples.

Animals↗