Evaluation of an immunoassay for direct detection of Escherichia coli O157 in stool specimens.

An enzyme-linked immunosorbent assay (ELISA) produced by LMD Laboratories, Inc., Carlsbad, Calif., was compared with culture for the detection of Escherichia coli O157. Nine of 185 stool specimens evaluated had positive results by the LMD E. coli O157 ELISA and grew E. coli O157 on culture; 174 had negative by LMD E. coli O157 ELISA results and did not grow E. coli O157 on culture. Of 174 specimens negative by LMD E. coli O157 ELISA, 117 specimens grew other enteric pathogens: Campylobacter spp. (46 isolates), Salmonella spp. (43 isolates), Yersinia spp. (20 isolates), and Shigella spp. (8 isolates). There were two indeterminant results by the LMD E. coli O157 ELISA. One stool specimen did not grow other enteric pathogens on culture, and one grew a Campylobacter sp. on culture. Both had negative LMD E. coli O157 ELISA results upon repeat testing. The LMD E. coli O157 ELISA is an accurate, easy-to-read screening method for the detection of E. coli O157 in fecal specimens.     

Simultaneous detection of six diarrhea-causing bacterial pathogens with an in-house PCR-luminex assay

Diarrhea can be caused by a range of pathogens, including several bacteria. Conventional diagnostic methods, such as culture, biochemical tests, and enzyme-linked immunosorbent assay (ELISA), are laborious. We developed a 7-plex PCR-Luminex assay to simultaneously screen for several of the major diarrhea-causing bacteria directly in fecal specimens, including pathogenic Aeromonas, Campylobacter jejuni, Campylobacter coli, Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia. We included an extrinsic control to verify extraction and amplification. The assay was first validated with reference strains or isolates and exhibited a limit of detection of 10(3) to 10(5) CFU/g of stool for each pathogen as well as quantitative detection up to 10(9) CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested for Campylobacter by ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% for Aeromonas, 89% and 93% for Campylobacter, 96% and 95% for Salmonella, 94% and 94% for Shigella, 92% and 97% for Vibrio, and 100% and 100% for Yersinia, respectively. All discrepant results were further examined by singleplex real-time PCR assays targeting different gene regions, which revealed 89% (55/62 results) concordance with the PCR-Luminex assay. The fluorescent signals obtained with this approach exhibited a statistically significant correlation with the cycle threshold (C(T)) values from the cognate real-time PCR assays (P < 0.05). This multiplex PCR-Luminex assay enables sensitive, specific, and quantitative detection of the major bacterial causes of gastroenteritis.     

Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.     

Evaluation of Statens Serum Institut Enteric Medium for Detection of Enteric Pathogens

The efficacy of the Statens Serum Institut (SSI) enteric medium for isolation and direct identification of enteric pathogens was evaluated. Six different biochemical reactions can be read by using the SSI enteric medium, allowing direct identification of a range of enteric pathogens. All 248 gram-negative bacterial species that were tested grew on the SSI enteric medium. Only 10 of 248 bacteria (4%) showed discrepant results in the biochemical reactions, and none of these were enteric pathogens. Forty-three of 47 enteric pathogens (92%) produced identical rates of semiquantitative growth on the SSI enteric medium and 5% blood agar, whereas three Vibrio spp. and one Aeromonas spp. showed reduced growth. Gram-positive bacteria did not grow on the SSI enteric medium. Most enteric pathogens had a detection limit of 50 bacteria per ml of feces, but higher numbers of Vibrio spp. and some Shigella spp. were required for detection. The growth rates of 125 enteric pathogens and 12 Yersinia spp. on the SSI enteric medium, xylose lysine deoxycholate (XLD), Hektoen enteric (HE), Salmonella-Shigella (SS), and cefsulodin-irgasan-novobiocin (CIN) agar were compared. Detection rates after application of 200 CFU were 99% for SSI enteric medium, 92% for XLD, 88% for HE, and 82% for SS agar. The 12 Yersinia spp. grew excellently on both the SSI enteric medium and CIN agar. We conclude that the performance of the SSI enteric medium compares favorably to those of other media tested. Its ability to detect Yersinia spp. may limit the number of media needed in the typical laboratory. The direct identification of enteric pathogens on the medium may also provide a more rapid diagnosis.     

Comparison of a shiga toxin enzyme-linked immunosorbent assay and two types of PCR for detection of shiga toxin-producing Escherichia coli in human stool specimens

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of sporadic cases of disease as well as serious outbreaks worldwide. The spectrum of illnesses includes mild nonbloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. STEC produces one or more Stxs, which are subdivided into two major classes, Stx1 and Stx2. The ingestion of contaminated food or water, person-to-person spread, and contact with animals are the major transmission modes. The infective dose of STEC may be less than 100 organisms. Effective prevention of infection is dependent on rapid detection of the causative bacterial pathogen. In the present study, we examined 295 stool specimens for the presence of Stx-producing E. coli by three different methods: an Stx enzyme-linked immunosorbent assay, a conventional PCR assay, and a LightCycler PCR (LC-PCR) assay protocol recently developed by our laboratory at the Institute of Medical Microbiology at Hannover Medical School. Our intent was to compare these three methods and to examine the utility of the STEC LC-PCR protocol in a clinical laboratory. The addition of a control DNA to each sample to clearly discriminate inhibited specimens from negative ones enhanced the accuracy of the LC-PCR protocol. From our results, it can be concluded that LC-PCR is a very useful tool for the rapid and safe detection of STEC in clinical samples.     

Development of a serotype-specific DNA microarray for identification of some Shigella and pathogenic Escherichia coli strains

Shigella and pathogenic Escherichia coli are major causes of human infectious diseases and are responsible for millions of cases of diarrhea worldwide every year. A convenient and rapid method to identify highly pathogenic serotypes of Shigella and E. coli is needed for large-scale epidemiologic study, timely clinical diagnosis, and reliable quarantine of the pathogens. In this study, a DNA microarray targeting O-serotype-specific genes was developed to detect 15 serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types 4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was tested against 186 representative strains of all Shigella and E. coli O serotypes, 38 clinical isolates, and 9 strains of other bacterial species that are commonly present in stool samples and was shown to be specific and reproducible. The detection sensitivity was 50 ng genomic DNA or 10(4) CFU per ml in mock stool specimens. This is the first report of a microarray for serotyping Shigella and pathogenic E. coli. The method has a number of advantages over traditional bacterial culture and antiserum agglutination methods and is promising for applications in basic microbiological research, clinical diagnosis, food safety, and epidemiological surveillance.     

Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.     

Detection of Salmonella spp. in clinical specimens by capture enzyme-linked immunosorbent assay.

A capture enzyme-linked immunosorbent assay (ELISA; Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was used to detect Salmonella spp. in clinical and artificially inoculated specimens. In patients with bacteremia caused by Salmonella spp., 48% (12 of 25) and 82% (13 of 16) of serum and urine specimens, respectively, were positive for Salmonella spp., as determined by ELISA. All serum and urine specimens collected from healthy individuals (25 specimens) or patients whose blood cultures grew gram-negative bacteria other than Salmonella spp. (18 specimens) were negative for Salmonella spp., as determined by ELISA. For blood culture bottles in which Salmonella spp. (16 specimens) was grown the ELISA was positive (100%), while it was negative for all the 65 blood culture bottles in which gram-negative bacteria other than Salmonella spp. (42 specimens) or gram-positive bacteria (23 specimens) were grown. All samples of urine (16 specimens), stool (8 specimens), serum (16 specimens), culture media (12 specimens), and blood culture bottles (reported sterile after 2 weeks of incubation; 16 specimens) that were artificially inoculated with 10(3) to 10(7) CFU of four species of Salmonella per ml were positive by ELISA. Similar specimens inoculated with or containing various species other than Salmonella were negative by this test. Thus, ELISA offers a promising opportunity for the rapid detection of Salmonella spp. in clinical microbiology laboratories.     

Rapid isolation of Yersinia spp. from feces.

Direct plating or cold enrichment or both have been used to isolate Yersinia spp. from feces. Freeze-shock double enrichment and KOH treatment have been recommended for recovery of Yersinia enterocolitica from surface waters and food, respectively. These techniques were evaluated as alternatives for rapid recovery of Yersinia spp. from feces. Stool samples were homogenized in buffered saline and autoclaved. Escherichia coli. Klebsiella pneumoniae, and Pseudomonas aeruginosa were each added to the suspension at a final concentration of 1.5 x 10(6) colony-forming units per ml. Yersinia cells were then added to a final concentration of 1.5 x 10(3), 1.5 x 10(4), 1.5 x 10(5), or 1.5 x 10(6) colony-forming units per ml. A total of 21 strains of Y. enterocolitica, 2 of Yersinia kristensenii, and 1 each of Yersinia intermedia and Yersinia fredriksenii were tested. For freeze-shock double enrichment, seeded stool samples were frozen overnight (-70 degrees C), transferred successively to m-tetrathionate broth (6 h. 37 degrees C) and selenite broth (2 h 37 degrees C), and plated on MacConkey, salmonella-shigella, and cellobiose-arginine-lysine agars for quantitation. For KOH treatment, seeded stool samples were mixed with 0.5% KOH at a ratio of 1:2 for 2 min and plated as described above. E. coli, K. pneumoniae, and P. aeruginosa were virtually eliminated after either method was used. All Yersinia strains were recovered after KOH treatment even at the lowest initial concentration (1.5 x 10(3) colony-forming units per ml). However, after freeze-shock double enrichment, not all strains were retrievable, and those isolates which were recovered were grown only from samples containing the highest number of Yersinia strains (1.5 x 10(6) colony-forming units per ml). KOH treatment of stool samples seems to be a viable substitute for more protracted methods of recovering Yersinia spp.     

Cadmium chloride susceptibility, a characteristic of Campylobacter spp.

We report a simple diagnostic characteristic useful in the presumptive identification of Campylobacter jejuni and Campylobacter coli. Filter paper disks impregnated with cadmium chloride were placed on streaked agar medium. Zones of growth inhibition for Campylobacter spp. occurred at 1.25 micrograms per disk. Other enteropathogens (Salmonella spp., Shigella spp., Vibrio cholerae, Vibrio parahaemolyticus, Escherichia coli, and Yersinia enterocolitica) were resistant to at least 40 micrograms per disk, with the exception of a strain of Shigella flexneri, which showed first susceptibility at 10 micrograms per disk. Most of the 52 Campylobacter strains, which were isolated from human clinical and animal sources, showed zones of inhibition greater than 10 mm with 2.5 micrograms of cadmium chloride per disk. At 20 micrograms per disk, Campylobacter isolates from clinical sources were significantly (P less than 0.01) more susceptible to cadmium chloride inhibition than were those from meat samples.     

Epidemiology of bacterial pathogens associated with infectious diarrhea in Djibouti.

During a survey examining the causes of diarrhea in the East African country of Djibouti, 140 bacterial pathogens were recovered from 209 diarrheal and 100 control stools. The following pathogens were isolated at comparable frequencies from both diarrheal and control stools: enteroadherent Escherichia coli (EAEC) (10.6 versus 13%), enterotoxigenic E. coli (ETEC) (11 versus 10%), enteropathogenic E. coli (EPEC) (7.7 versus 12%), Salmonella spp. (2.9 versus 3%), and Campylobacter jejuni-C. coli (3.3 versus 5%). Surprisingly, the EAEC strains isolated did not correspond to well-recognized EPEC serogroups. No Yersinia spp., enteroinvasive E. coli, or enterohemorrhagic E. coli were isolated during the course of this study. Only the following two genera were recovered from diarrheal stools exclusively: Shigella spp. (7.7%) and Aeromonas hydrophila group organisms (3.3%). Shigella flexneri was the most common Shigella species isolated. Patients with Shigella species were of a higher average age than were controls (27 versus 13 years), while subjects with Campylobacter or Salmonella species belonged to younger age groups (2.6 and 1.6 years, respectively). Salmonella cases were more often in females. Shigella diarrhea was associated with fecal blood or mucus and leukocytes. ETEC was not associated with nausea or vomiting. Anorexia, weight loss, and fever were associated with the isolation of Salmonella and Aeromonas species. EAEC, ETEC, EPEC, and Shigella species were resistant to most drugs used for treating diarrhea in Africa, while the antibiotic most active against all bacteria tested was norfloxacin. We conclude that in Djibouti in 1989, Shigella and Aeromonas species must be considered as potential pathogens whenever they are isolated from diarrheal stools and that norfloxacin should be considered the drug of choice in adults for treating severe shigellosis and for diarrhea prophylaxis in travelers.     

Evaluation of a rapid method to exclude the presence of certain enteric pathogens in stool specimens

A new commercial method intended to exclude the presence of Salmonella spp., Shigella spp., and Yersinia enterocolitica and to presumptively identify Salmonella isolates within 2 h after primary isolation from stool specimens was evaluated. This system is marketed in Europe as API Z and in the United States as Rapid SST. The strip consists of five pairs of cupules for the screening of five lactose-negative colonies. The first cupule of each pair detects the presence of five enzymatic activities, whereas the second serves to maintain the strain for additional testing if necessary. A total of 197 fresh isolates from stool specimens and 217 stock cultures of Salmonella spp., Shigella spp., and Yersinia enterocolitica were tested, with the API 20E system as a reference method. In the stool specimens, 77.3% of the bacteria could be excluded from further workup for the presence of these organisms within 2 h. Over 97% of the stock strains and each of three fresh Salmonella isolates tested produced a reaction pattern corresponding to a correct presumptive identification. This reaction pattern was not produced by any isolate other than the Salmonella isolates. The API Z system can be used as a screen for the presence of Salmonella and Shigella spp. and can provide an accurate presumptive identification of Salmonella isolates within 2 h after primary isolation.     

Two-year survey of etiologic agents of diarrheal disease at San Lazaro Hospital, Manila, Republic of the Philippines.

The prevalence of bacterial pathogens and rotavirus in 2,908 patients with diarrhea who were admitted to San Lazaro Hospital in Manila in 1983 and 1984 was determined. One or more enteric pathogens were isolated or detected in samples from 1,698 (58.4%) patients. Isolation rates for the various enteropathogens were as follows: rotavirus, 30.6%; Shigella spp., 11.6%; Salmonella spp., 9.2%; enterotoxigenic Escherichia coli (1983 only), 7.8%; Vibrio cholerae biotype eltor, 3.8%; non-O1 V. cholerae, 2.8%; Vibrio parahaemolyticus, 1.7%; other Vibrio spp., 1.1%; Campylobacter jejuni, 3.0%; Aeromonas hydrophila, 1.3%; and Plesiomonas shigelloides 1.1%. Giardia lamblia and Entamoeba histolytica were detected in 0.6 and 0.1%, respectively, of stool samples examined. Determination of the etiologic role of isolates was complicated by one or more of the following factors: isolation of multiple enteric pathogens (302 cases); isolation of Salmonella spp., enterotoxigenic E. coli, and C. jejuni from a similar proportion of asymptomatic control patients and patients with diarrhea; and isolation of a high proportion of certain pathogens (especially Salmonella spp.) only from enrichment broth, suggesting infection with a small number of organisms. Isolation of V. cholerae eltor was seasonal, with the majority of cases occurring in the rainy months. In addition, the number of patients with diarrhea increased with the onset of the monsoon rains and peaked during the months of maximum rainfall. Rotavirus infection occurred in both children and adults throughout the year and was the most frequently identified cause of diarrhea in children under 5 years of age. Shigella spp. were the most common agents of diarrhea in adults.     

Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis

The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibrio spp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx₁ and stx₂) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.     

Evaluation of Luminex xTAG Gastrointestinal Pathogen Analyte-Specific Reagents for High-Throughput,Simultaneous Detection of Bacteria,Viruses, and Parasites of Clinical and Public Health Importance

Acute diarrheal disease (ADD) can be caused by a range of pathogens, including bacteria, viruses, and parasites. Conventional diagnostic methods, such as culture, microscopy, biochemical assays, and enzyme-linked immunosorbent assays (ELISA), are laborious and time-consuming and lack sensitivity. Combined, the array of tests performed on a single specimen can increase the turnaround time (TAT) significantly. We validated a 19plex laboratory-developed gastrointestinal pathogen panel (GPP) using Luminex xTAG analyte-specific reagents (ASRs) to simultaneously screen directly in fecal specimens for diarrhea-causing pathogens, including bacteria (Campylobacter jejuni, Salmonella spp., Shigella spp., enterotoxigenic Escherichia coli [ETEC], Shiga toxin-producing E. coli [STEC], E. coli O157:H7, Vibrio cholerae, Yersinia enterocolitica, and toxigenic Clostridium difficile), parasites (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica), and viruses (norovirus GI and GII, adenovirus 40/41, and rotavirus A). Performance characteristics of GPP ASRs were determined using 48 reference isolates and 254 clinical specimens. Stool specimens from individuals with diarrhea were tested for pathogens using conventional and molecular methods. Using the predictive methods as standards, the sensitivities of the GPP ASRs were 100% for adenovirus 40/41, norovirus, rotavirus A, Vibrio cholerae, Yersinia enterocolitica, Entamoeba histolytica, Cryptosporidium spp., and E. coli O157:H7; 95% for Giardia lamblia; 94% for ETEC and STEC; 93% for Shigella spp.; 92% for Salmonella spp.; 91% for C. difficile A/B toxins; and 90% for Campylobacter jejuni. The overall comparative performance of the GPP ASRs with conventional methods in clinical samples was 94.5% (range, 90% to 97%), with 99% (99.0% to 99.9%) specificity. Implementation of the GPP ASRs enables our public health laboratory to offer highly sensitive and specific screening and identification of the major ADD-causing pathogens.     

Development and testing of invasion-associated DNA probes for detection of Shigella spp. and enteroinvasive Escherichia coli.

Genetic determinants of the invasive phenotype of Shigella spp. and enteroinvasive Escherichia coli (EIEC), two common agents of bacillary dysentery, are encoded on large (180- to 210 kilobase), nonconjugative plasmids. Several plasmid-encoded antigens have been implicated as important bacterial ligands that mediate the attachment and invasion of colonic epithelial cells by the bacteria. Selected invasion plasmid antigen (ipa) genes have recently been cloned from Shigella flexneri serotype 5 into the lambda gt11 expression vector. Portions of three ipa genes (ipaB, ipaC, and ipaD) were tested as DNA probes for diagnostic detection of bacillary dysentery. Under stringent DNA hybridization conditions, all three DNA sequences hybridized to a single 4.6-kilobase HindIII fragment of the invasion plasmids of representative virulent Shigella spp. and EIEC strains. No hybridization was detected in isogenic, noninvasive Shigella mutants which had lost the invasion plasmid or had deleted the ipa gene region. Furthermore, these probes did not react with over 300 other enteric and nonenteric gram-negative bacteria tested, including Salmonella, Yersinia, Edwardsiella, Campylobacter, Vibrio, Klebsiella, Aeromonas, Enterobacter, Rickettsia, and Citrobacter spp. and various pathogenic E. coli strains. The use of unique invasion-essential gene segments as probes for the specific detection of invasive dysentery organisms should benefit both epidemiologic and diagnostic analyses of Shigella spp. and EIEC.     

2010年广州某医疗中心儿童夏季细菌性腹泻的病原学调查

目的研究广州地区小儿夏季细菌性腹泻的病原菌分布。方法采集2010年5～7月广州市妇女儿童医疗中心珠江新城院区腹泻患儿的大便标本进行常规病原菌的分离培养,通过生化反应和血清凝集试验进行鉴定和分型,并使用金标法对空肠弯曲菌抗原进行检测。结果从110份标本中检出44株病原菌,检出率为40.0%。其中致病性大肠埃希菌17株,2岁以下患儿检出15株;空肠弯曲菌12株,2岁以下患儿检出10株;沙门菌6株;念珠菌纯生长6株;产气荚膜杆菌3株。结论广州地区夏季儿童细菌性腹泻的主要病原菌是致病性大肠埃希菌及空肠弯曲菌,两者的易感人群以2岁以下婴幼儿为主。     

Use of an alkaline phosphatase-labeled synthetic oligonucleotide probe for detection of Campylobacter jejuni and Campylobacter coli.

A commercially available synthetic nucleic acid probe (SNAP) conjugated to alkaline phosphatase was compared with standard culture techniques for detecting Campylobacter species. The SNAP was able to detect either 5 ng of C. jejuni DNA or 10(5) CFU of bacteria. The SNAP could also detect DNA extracted from 10(5) CFU in mock-infected stool samples. The SNAP detected C. jejuni and C. coli but showed no reactivity with C. laridis, C. fetus subsp. fetus, C. fetus subsp. venerealis, C. fennelliae, "C. upsaliensis," C. cinaedii, C. fecalis, C. hyointestinalis, C. mucosalis, or Helicobacter (Campylobacter) pylori. The SNAP also showed no cross-reactivity with other enteric pathogens. When applied to pure cultures, the SNAP detected 55 clinical isolates of C. jejuni and 11 clinical isolates of C. coli, with an accuracy of 100%. When applied directly to clinical specimens, the SNAP detected Campylobacter spp. in 19 of 23 culture-positive stool specimens (sensitivity, 82.6%; specificity, 100%). Pure cultures of the Campylobacter strains isolated from the four probe-negative, culture-positive stool specimens gave positive reactions with the SNAP. While the SNAP had excellent sensitivity and specificity for isolated bacterial colony isolates, the main limitation to the Campylobacter probe detection kit may be the sensitivity limit on direct detection of Campylobacter organisms in stools.     

Real-time fluorescence PCR assays for detection and characterization of heat-labile I and heat-stable I enterotoxin genes from enterotoxigenic Escherichia coli

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC.     

Differentiation of Giardia duodenalis from other Giardia spp. by using polymerase chain reaction and gene probes.

Giardia spp. are waterborne organisms that are the most commonly identified pathogenic intestinal protozoans in the United States. Current detection techniques for Giardia species in water include microscopy and immunofluorescence techniques. Species of the genus Giardia are classified on the basis of taxonomic criteria, such as cell morphology, and on host specificity. We have developed a polymerase chain reaction- and gene probe-based detection system specific for Giardia spp., which can discriminate between the relevant species of the G. duodenalis type pathogenic to humans and other Giardia species that are not human pathogens. This method can detect a single Giardia cyst and is therefore sensitive enough for environmental monitoring.