A multiple protocol to improve diagnosis and isolation of Shiga toxin-producing Escherichia coli from human stool specimens

Many infections caused by Shiga toxin-producing Escherichia coli (STEC) are undiagnosed, particularly non-O157 STEC. We evaluated the use of a multiple protocol approach to improve diagnosis, isolation, and characterization of STEC strains. Among 18 presumptive STEC-positive stool samples received by the INOVA Fairfax Hospital, Falls Church, VA, in 2006, 16 were Shiga toxin positive. From these 16 stool samples, 8 O157:H7 and 5 non-O157 STEC were isolated by plating onto sorbitol MacConkey (SMAC) agar. The remaining 5 stool samples that did not yield colonies on SMAC agar plates were enriched. All enriched samples were Shiga toxin positive, and 2 O157:H7 and 1 non-O157 STEC were subsequently isolated. The 2 remaining enriched samples did not yield isolates; however, based on polymerase chain reaction (PCR) analysis, both samples contained STEC genes. Based on PCR analysis of non-O157 strains, 3 strain types were identified. Samples from 3 patients, received within 2 days of one another, had a similar gene profile-eae and stx(1) negative and stx(2) positive-suggesting that these patients were likely infected with the same strain. Our results indicate that a multiple protocol approach is necessary to reliably diagnose and isolate STEC strains, and that PCR profiling of strains could allow for more rapid identification of outbreaks.     

Real-time PCR detection of Mycoplasma pneumoniae in respiratory specimens

A real-time PCR assay targeting the phosphotransferase system I gene (ptsI) of Mycoplasma pneumoniae was compared to 2 commercially available PCR assays targeting the P1 cytadhesion gene (the LightMix®Kit Mycoplasma pneumoniae [TIB MOLBIOL, Adelphia, NJ, USA] and M. pneumoniae Analyte Specific Reagent [Focus Diagnostics, Cypress, CA, USA] assays) and to a PCR assay targeting the M. pneumoniae repetitive element, RepMP1. Thirty previously positive specimens including 15 throat swab, 10 bronchoalveolar lavage, and 5 sputum specimens, all tested positive with the ptsI and M. pneumoniae ASR assays. Among the previously positive specimens, 14/15 throat swab, 9/10 bronchoalveolar lavage, and 4/5 sputum specimens tested positive with the LightMix®Kit Mycoplasma pneumoniae assay and 13/15 throat swab, 10/10 bronchoalveolar lavage, and 4/5 sputum specimens tested positive with the RepMP1 assay. Forty previously negative clinical specimens tested negative using the ptsI assay. A PCR assay targeting M. pneumoniae ptsI performs equivalently to assays targeting the P1 cytadhesion gene or RepMP1.     

Preliminary evaluation of a procedure for improved detection of Shiga toxin-producing Escherichia coli in fecal specimens

Culture confirmation of Shiga toxin-producing Escherichia coli (STEC) is very important for epidemiologic analysis. However, isolation of non-O157 STEC on conventional selective media such as sorbitol–MacConkey agar (SMAC) can be difficult because of heavy growth of competing bacteria and its phenotypical similarity to commensal nonpathogenic E. coli. An acid enrichment procedure was introduced in this study to facilitate detection of STEC from patients who were symptomatic. Forty-seven clinical fecal broths, which tested positive for Shiga toxin by commercial immunoassay, were processed for the isolation of STEC by both conventional and the acid enrichment methods. The acid enrichment method and conventional culture recovered STEC from 91% (43/47) and 70% (33/47) of the fecal broths, respectively. Neither method retrieved STEC in 3 specimens. Thirty-six STEC were successfully serogrouped, which included O26 (n = 11), O157 (n = 9), O103 (n = 7), O121 (n = 3), O111 (n = 2 each), O28AC, O146, O76, and O undetermined (n = 1 each). The analysis of STEC isolates by real-time PCR indicated that all 9 E. coli O157 contained stx2 gene alone or in combination with stx1. Non-O157 STEC more frequently contained stx1 only, and about one-third possessed stx2. The novel acid enrichment protocol greatly reduced the growth of competitor colonies on RTN and TCSMAC. The study demonstrated that incorporation of an acid enrichment procedure in clinical testing improved the isolation of STEC in fecal specimens.     

Comparison of Shiga Toxin-Producing Escherichia coli Detection Methods Using Clinical Stool Samples

Molecular diagnostic tools capable of identifying Shiga toxin-specific genetic determinants in stool specimens permit an unbiased approach to detect Shiga toxin-producing Escherichia coli (STEC) in clinical samples and can indicate when culture-based isolation methods are required. It is increasingly recognized that clinically relevant STEC are not limited to the singular O157 serotypes, and therefore diagnostic assays targeting toxin-encoding determinants must be able to account for any genetic variation that exists between serotypes. In this study conventional PCR and four real-time PCR assays (HybProbe, TaqMan, SYBR Green, and LUX) targeting the stx1 and stx2 Shiga toxin coding sequences were used to identify STEC in enriched stool samples (n = 36) and a panel of O157 and non-O157 strains (n = 64). PCR assays targeting stx1 and stx2 had variable specificity and sensitivity values with enriched stool samples. Molecular assays using DNA from pure cultures revealed that some primers were not sensitive to all stx2 variants. This evaluation concluded that the TaqMan-based probes were most appropriate in high throughput clinical diagnostic laboratories in consideration of cost, turn around time, and assay performance.Shiga toxin-producing Escherichia coli (STEC) that belong to serotype O157:H7 are recognized globally as important causative agents of enterocolitis food poisoning. Several prominent outbreaks of this STEC serotype have also gained significant public health attention, leading to the overall appreciation of O157:H7 as a major public health concern in the food production industry.^1,2,3 STEC can lead to a wide array of clinical manifestations ranging from barely noticeable acute gastritis to bloody diarrhea, and occasionally to complications such as hemolytic uremic syndrome (HUS).^4,5,6 These infections, hospitalizations, and subsequent complications have been shown to result in dramatic costs to both the health care system and the state infection control departments involved in their management.⁷ Typically, STEC infection is most severe in pediatric and elderly patients, whereas adults do not present with severe symptoms in most cases.⁸ Additionally, Shiga toxins, encoded by stx1 and stx2, are the primary virulence factor possessed by STEC and contribute to the manifestation of both bloody diarrhea and HUS.⁵ Although a great deal of attention (both in the general public and the scientific communities) has been focused on the O157:H7 serotype, several studies suggest that up to 50% of STEC illness is caused by serotypes other than O157^{9,10,11,12,13} of which there are over 100.¹⁴O157 STEC are routinely screened for in the clinical microbiology laboratory by selective plating on sorbitol MacConkey media, which exploits the nonsorbitol fermenting phenotype of most O157 strains. As a result, O157 can readily be distinguished from normal E. coli flora. However, the recent identification of sorbitol fermenting O157 strains associated with HUS calls for additional screening methods to be used in addition to sorbitol MacConkey.¹⁵ BBL CHROMagar O157 (BD Canada, Mississauga, ON, Canada) plated medium is an alternative method for identifying O157 growth based on differential colorimetric colony growth.¹⁶ However, these plating methods only select for sorbitol fermenting strains of O157 or simply O157 strains of STEC, respectively, whereas commensal E. coli and non-O157 STEC are indistinguishable on both media and therefore require additional molecular diagnostic assays. To date, no widely used standard method for identifying non-O157 STEC infections is used by clinical diagnostic laboratories.There are several research methods reported for the detection of non-O157 STEC, most of which focus on the detection of Shiga toxins or Shiga toxin-encoding genetic determinants. A standardized method for detecting the presence of toxin in stool involves filtration of stool for purification of toxin with subsequent incubation with Vero tissue culture cells. This assay requires a highly skilled technician to maintain the cell lines as well as monoclonal antibodies for confirmation of Shiga toxin. More importantly, the Vero cell assay is considered time and labor intensive, not for practical clinical diagnostic purposes. However, the Meridian Premier EHEC kit (Meridian Diagnostics, Inc., Cincinnati, OH) can detect Shiga toxin in stool samples via an immunoassay and can aid in the identification of non-O157 STEC. Alternatively, a myriad of PCR-based assays designed to detect stx1 and stx2 in E. coli has been described.^{6,17,18,19,20,21,22,23,24,25,26} Recently, the Centers for Disease Control and Prevention released updated guidelines for the detection of STEC in relation to acute community-acquired diarrhea, which included specific testing for Shiga toxins or their genetic determinants in addition to traditional culture (http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5812a1.htm, updated Oct 16, 2009).We compared four real-time PCR assays (two described in this study) as well as a conventional PCR method for the detection of STEC stx1 and stx2 genes. The assays were compared for sensitivity, specificity, detection threshold, as well as cost and time requirements. These methods were compared for their ability to detect various STEC serotypes from pure cultures as well as from stool specimens and optimized for the respective amplification platforms located in four different laboratories. Extraction of DNA from cultures or spiked stool samples was performed in one selective laboratory and distributed to the other laboratories for testing. The results of this study provide guidelines for diagnostics laboratories to adopt a methodology according to available platform and budget.     

Molecular diagnosis of Arcobacter and Campylobacter in diarrhoeal samples among Portuguese patients

The present study was conducted to investigate the prevalence and diversity of Arcobacter and Campylobacter spp. in 298 stool samples of patients with diarrhoea, collected from 22 Portuguese hospitals, between September and November 2012. Detection of Arcobacter and Campylobacter spp. was performed using molecular-based detection techniques, such as real-time fluorescence resonance energy transfer PCR, species-specific PCR, and sequencing of amplified PCR products. Overall, 1.3% of the samples were positive for Arcobacter butzleri and 0.3% for Arcobacter cryaerophilus. Campylobacter spp. were found in 31.9% of diarrhoeic faeces. Campylobacter jejuni and Campylobacter concisus were the most prevalent species (13.7% and 8.0%, respectively). The prevalence of Arcobacter and Campylobacter spp. was significantly different between children and adults (39.7% versus 22.8%, P = 0.003). We underline the high prevalence of these pathogens in diarrhoeal samples among Portuguese patients, with particular relevance in the paediatric age group.     

Semi-automated fluorogenic PCR assays (TaqMan) forrapid detection of Escherichia coli O157:H7 and other shiga toxigenic E. coli.

Semi-automated detection of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC) was achieved using fluorogenic polymerase chain reaction (PCR). These PCR assays were designed to amplify 80, 120 and 150 bp regions of virulence genes stx1, stx2 and eaeA, respectively, using specific primers. The fluorogenic probes were used for specific detection of amplified products of the stx1 and stx2 genes of STEC, and the eaeA gene of EHEC O157:H7. For multiplex PCR assay, the three sets of primers and fluorogenic probes were included in one reaction to simultaneously amplify and detect any of the three targeted virulence genes. In non-multiplex PCR assay, each of the three virulence genes was amplified and detected in independent reactions. The specificity of these assays was evaluated using suspensions of STEC and other bacterial species lacking stx1, stx2 and eaeA. The multiplex assay detected all STEC harbouring any combination of three virulence genes. Three non-multiplex PCR reactions identified types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-O157:H7 STEC. Sensitivity limits of these assays in beef and faeces inoculated with EHEC O157:H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively. These assays can be completed within 8-10 h when performed simultaneously or within 13 h if the multiplex assay is used as an initial screen for detecting STEC and the non-multiplex assay is used for subsequent detection of stx1 and stx2 of STEC and eaeA of EHEC O157:H7 Copyright 1999 Academic Press.     

Optimization of a 3'-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR

Enterohemorrhagic Escherichia coli are harmful human pathogens capable of causing bloody diarrhea and vomiting. An important serotype commonly associated with human illness is the E. coli O157:H7 serotype. Unlike other real-time polymerase chain reaction (PCR) methods for identifying E. coli O157:H7, this study describes the development and optimization of a real-time PCR method targeting a conserved point mutation at +93 in the uidA (gusA) gene that is unique to O157:H7, distinguishing it from non-O157:H7 serotypes. A TET-labeled Minor Groove Binder (MGB) DNA probe was designed for use in a 5' nuclease PCR assay. Using a panel of two E. coli O157:H7 strains, three E. coli non-O157:H7 strains, and one non-E. coli species, the assay was optimized for the specific detection of the E. coli O157:H7 strains. Optimal conditions were identified at high anneal/extend temperatures, low magnesium concentrations, and low probe concentrations, resulting in correct identification of E. coli O157:H7 and non-O157:H7 strains. The improved specificity of MGB probes for single base pair mismatches such as the +93 uidA mutation provides a novel approach towards rapid identification of E. coli O157:H7.     

江苏省东台地区家畜非O157产志贺毒素大肠埃希菌耐药性及多位点序列分型分析

  目的  了解江苏省东台地区家畜来源非O157产志贺毒素大肠埃希菌（STEC）抗生素耐药表型、耐药基因,以及多位点序列分型（MLST）情况。  方法  于2019年5月,江苏省东台市采集家畜粪便样本301份（羊粪便231份,牛粪便70份）,分离非O157 STEC菌株。 采用改良微量肉汤法测定菌株对21种抗生素最小抑菌浓度（MIC）;通过全基因组测序技术对O∶H血清型、耐药基因和多位点序列型别进行预测。  结果  在68株非O157 STEC中,有32株对至少1种药物耐药。 其中,STEC对四环素耐药率最高（42.6%）,其次分别对阿奇霉素（36.8%）、复方新诺明（35.3%）、链霉素（32.3%）、氯霉素（30.9%）、环丙沙星（29.4%）等9种抗生素耐药;共识别出25种耐药基因,其中,氨基糖苷类耐药基因和叶酸途径抑制剂类耐药基因携带率最高（44.1%）,其次为四环素类耐药基因tet(A)（42.6%）;MLST将分离株分为13种ST型,其中ST43（19.1%）和ST155（16.2%）比例较高。 最小生成树显示,具有相同血清型的STEC菌株多聚集在一起。 STEC中4种ST型别（ST25、ST40、ST43、ST675）分别与引起肠溶血性尿毒综合征相关的肠出血性大肠埃希菌（HUSEC）聚类成簇。  结论  江苏东台地区家畜中非O157 STEC耐药形势复杂,且存在多重耐药现象。 此外,该地区STEC菌株对人群存在潜在威胁,家畜作为非O157 STEC宿主应引起重视。     

Detection and characterization of thefimAgene ofEscherichia coliO157:H7

An expected 850-bp DNA fragment containingfimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains ofEscherichia coliusing the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13HinPI and fourSau96I restriction profiles among these 39E. colistrains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared byE. coliO157:H7, O157:H^- and a few O55 serotype strains. DNA sequence analysis of thefimAregion demonstrated thatE. coliO157:H7 strain 933 and O157:H⁻strain E32511 contained identical DNA sequences that were distinct from otherE. colistrains, especially a 16-bp sequence 5′ tofimAthat was conspicuously absent only inE. coliO157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from allE. coliO157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detectE. colistrains of the O157:H7 serotype.     

Evaluation of the EasyScreen™ Enteric Parasite Detection Kit for the detection of Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex,and Giardia intestinalis from clinical stool samples

The aim of this study was to evaluate the EasyScreen™ Enteric Parasite Detection Kit (Genetic Signatures, Sydney, Australia) for the detection and identification of 5 common enteric parasites: Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis in human clinical samples. A total of 358 faecal samples were included in the study. When compared to real-time PCR and microscopy, the EasyScreen™ Enteric Parasite Detection Kit exhibited 92–100% sensitivity and 100% specificity and detected all commonly found genotypes and subtypes of clinically important human parasites. No cross reactivity was detected in stool samples containing various other bacterial, viral, and/or protozoan species. The EasyScreen™ PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the 5 most important diarrhoea-causing enteric parasites that infect humans. It should be noted, however, that the EasyScreen™ Kit does not substitute for microscopy or for additional PCRs as it does not detect the pathogenic Coccidia spp. Cystoisospora belli or Cyclospora cayetanensis and it does not differentiate between pathogenic and nonpathogenic Entamoeba spp. This study also highlights the lack of sensitivity demonstrated by microscopy; as such, molecular methods should be considered the diagnostic method of choice for enteric parasites.     

Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia

A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10⁵ DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions.     

非O157产志贺毒素大肠杆菌分离株的志贺毒素基因变种及黏附相关基因分析

目的 了解我国部分地区非O157产志贺毒素大肠杆菌分离株的志贺毒素基因变种及其黏附相关基因,为进一步研究致病机制提供依据。 方法 采用聚合酶链反应(polymerase chain reaction,PCR)方法对29株分离菌株的stx1、stx2基因全长扩增并测序,通过与GenBank中已公布的变种序列比对确定菌株stx1、stx2基因的变种类型。对位于LEE毒力岛上的eaeA、escF、escC、tir、espA、espB、espD基因及LEE以外其他黏附相关基因iha、toxB、efa1、sfpA、lpfAO157/OI-141、 lpfAO157/OI-154、saa、lpfAO113、eibG进行PCR检测。 结果 25株stx1阳性菌株中有13株携带stx1a原毒素,12株携带stx1c变种;10株stx2阳性菌株中,7株携带stx2d变种,1株为stx2a原毒素,1株携带stx2g变种,1株携带与stx2e A亚单位、stx2d B亚单位最接近的stx2变种。LEE岛上的7个基因检测结果均为阴性,黏附相关基因iha阳性率为89.7%(26/29),saa阳性菌株3株、eibG阳性菌株1株,其余6个黏附相关基因均为阴性。 结论 我国部分非O157 STEC菌株的志贺毒素基因以stx1c、stx2d变种为主,LEE毒力岛不存在,而黏附相关基因iha广泛存在于不同血清型的产志贺毒素大肠杆菌菌株中。     

Detection of stx1, stx2, and eae genes of enterohemorrhagic Escherichia coli using SYBR Green in a real-time polymerase chain reaction

We report a multiplex real-time polymerase chain reaction method for detecting enterohemorrhagic Escherichia coli (EHEC) from strains or stool specimens. This assay detected the virulence genes stx1, stx2, and eae, without the use of probes. The method, which was validated on a collection of 143 EHEC strains, is simple, rapid, cost-effective, and sensitive.     

非O157产志贺毒素大肠埃希菌分离株血清型鉴定及主要毒力基因分析

目的 了解中国不同来源非O157产志贺毒素大肠埃希菌分离株血清型及主要毒力基因的流行情况。方法 采用O抗原基因簇特异性聚合酶链反应（PCR）结合全套O抗血清凝聚法确定O血清群,以PCR扩增和测序fliC基因确定H型;采用PCR方法对所有菌株进行stx1、stx2、eae及ehxA基因检测。结果 434株非O157 STEC分离株中,除不可分型菌株外,共检测出82种O血清群和28种H型,其中O20:H30、O2:H32和O2:H45为优势血清型。stx1、stx2和stx1+stx2 3种志贺毒素基因型的检出率分别为25.35%、64.98%和9.68%,而eae和ehxA基因的阳性率分别为3.92%和32.95%。仅15株菌同时携带3种毒力基因,且主要为血清型O26:H11的腹泻患者分离株。结论 我国非O157 STEC分离株血清型复杂多样,同时检测eae和ehxA基因对于发现高致病潜力的菌株具有重要参考价值。     

Prevalence of Bordetella pertussis and Bordetella parapertussis in Infants Presenting to the Emergency Department with Bronchiolitis

Background: The clinical presentation of Bordetella pertussis can overlap with that of respiratory syncytial virus (RSV), and coinfection does occur, but management differs. Hypothesis: The prevalence of B. pertussis is < 2% among Emergency Department (ED) patients with bronchiolitis. Our secondary hypothesis was that the prevalence of Bordetella parapertussis is also < 2% among these patients. Methods: Nasal washings were obtained from children up to 18 months of age (inclusive) who presented to a county hospital ED with a clinical diagnosis of bronchiolitis. These washings were frozen to −70°C before testing for B. pertussis and B. parapertussis using species-specific real-time polymerase chain reaction (PCR) assays. The assays were optimized to target conserved regions within a complement gene and the CarB gene, respectively. A Bordetella spp. genus-specific real-time PCR assay was designed to detect the Bhur gene of B. pertussis, B. parapertussis, and B. bronchiseptica. RSV antigen detection was also performed. Results: There were 227 patients enrolled. After exclusions, 204 remained in the analysis. RSV antigen testing was positive in 109/186 (59%) of the patients in whom it was performed. All samples were tested for B. pertussis. B. parapertussis testing could not be completed on 23 samples. No cases (0/204; 95% confidence interval [CI] 0–1.8%) tested positive for B. pertussis or B. parapertussis (0/181; 95% CI 0–2%). Conclusion: The prevalence of B. pertussis in children presenting to the ED with bronchiolitis was < 2%.     

Validation of the EntericBio Panel II® multiplex polymerase chain reaction system for detection of Campylobacter spp., Salmonella spp., Shigella spp., and verotoxigenic E. coli for use in a clinical diagnostic setting

A total of 717 faeces samples were tested prospectively using the EntericBio Panel II® detection system (Serosep, Limerick, Ireland), in parallel with routine laboratory testing, which combines the EntericBio® system with retrospective culture of each specimen where a target is detected. Discrepancy analysis was conducted using molecular methods. The EntericBio Panel II® assay produced 585 negative and 132 positive results, namely, Campylobacter spp. (n = 66); SLT 1 and/or SLT 2 (n = 64); Salmonella spp. (n = 5); and Shigella spp. (n = 0). Three samples were positive for more than 1 target. Of these results, 4 Campylobacter spp. detections and 4 SLT 1/ SLT 2 detections remained unconfirmed, and the system failed to detect 2 Campylobacter spp. targets detected by routine laboratory detection. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were calculated to be 98.4%, 98.7%, 93.9%, 99.7%, and 98.6%, respectively.     

Simplified protocol for DNA extraction and amplification of 2 molecular markers to detect and type Giardia duodenalis

We evaluated the ability of 3 kits: QIAmp® DNA stool mini kit (Qiagen, Hilden, Germany), PureLink PCR Purification®, and PureLink™ Genomic DNA® (Invitrogen, Carlsbad, CA, USA) for DNA extraction, and of 2 molecular markers (heat shock protein [HSP] and β-giardin genes) for detection and genotyping of Giardia duodenalis stool samples. The detection and typing limits of the markers were determined by the DNA concentration of trophozoites and cysts and were tested in 26 clinical samples. Of the 3 kits tested, the PureLink PCR Purification gave the best results when tested with clinical samples with low, intermediate, and high numbers of cysts. The DNA extracted from trophozoites and cysts was diluted successively in 1:2 ratios until it was no longer possible to observe the amplified product in polyacrylamide gel. Similarly, a suspension of cysts was diluted until no cysts were observed, and then the DNA was extracted. The amount of DNA of trophozoites and cysts for the typing of the parasite was smaller for the HSP marker than for β-giardin. Combined use of both markers allowed us to detect DNA of Giardia in parasitologically positive samples in a higher percentage (75%) than the results obtained for each marker and in 1 parasitologically negative sample, indicating that this combination increased the potential to accurately detect and genotype this parasite. We also concluded that the HSP marker has a higher limit of detection and typing than the β-giardin marker and that the DNA extraction method tested for G. duodenalis is simpler and more efficient than those that are currently in use and can be applied on a large scale.     

Co-colonization of vanA and vanB Enterococcus faecium of clonal complex 17 in a patient with bacteremia due to vanA E. faecium

A 53-year-old Vietnamese man with liver cirrhosis was transferred from a Vietnamese hospital to our tertiary care hospital in Korea in order to undergo a liver transplantation. Bacteremia due to vanA Enterococcus faecium was diagnosed, and stool surveillance cultures for vancomycin-resistant enterococci (VRE) were positive for both vanA and vanB E. faecium. Pulsed-field gel electrophoresis analysis revealed that the 2 vanA VRE isolates from the blood and stool were clonal, but the vanB VRE was unrelated to the vanA VRE. vanA and vanB VRE were ST64 and ST18, single-allele variations of clonal complex 17, respectively. This is the first case report of vanA VRE bacteremia in a Vietnamese patient and demonstrates the reemergence of vanB VRE since a single outbreak occurred 15 years ago in Korea. The reemergence of vanB VRE emphasizes the importance of VRE genotyping to prevent the spread of new VRE strains.     

Laboratory investigation of an Escherichia coli O157:H7 strain possessing a vtx2c gene with an IS1203 variant insertion sequence isolated from an asymptomatic food handler in Japan

We isolated an Escherichia coli O157:H7 strain that was negative for verocytotoxin production, but positive for the vtx2 gene using commercial kits, from an asymptomatic food handler. The laboratory investigations revealed that a 1310-bp insertion sequence, IS1203 variant, was present in the B subunit-coding region of the vtx2c gene.     

A new chromogenic agar medium,chromID VRE,to screen for vancomycin-resistant Enterococcus faecium and Enterococcus faecalis

We compared the performance of a chromogenic agar medium chromID VRE (bioMérieux, Marcy-l'Etoile, France) designed to recover and identify vancomycin-resistant enterococci (VRE) from clinical specimens with bile esculin azide vancomycin (BEAV) agar. For this study, 120 stool specimens were plated on chromID VRE and BEAV and examined after 24 and 48 h. At 24 h, the sensitivity and specificity were as follows: BEAV, 90.2% and 73%, respectively; chromID VRE, 86.3% and 100.0%, respectively. Furthermore, we determined that the sensitivity and specificity of chromID VRE for Enterococcus faecium were 85.4% and 100%, respectively, and for Enterococcus faecalis, 90% and 100%, respectively. We conclude that chromID VRE provides an equivalent sensitivity for the recovery of VRE from stool specimens, with improved specificity, and the added advantage of providing differentiation between vancomycin-resistant E. faecium and E. faecalis.