Detection of Shiga Toxin-Producing Escherichia coli by in Stitu hybridization and sequence Analysis of Stx2 / 대한임상미생물학회지

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) was found in several serotypes of E coli including 0157 serotype. Sorbitol-MacConkey agar: may be useful for the detection of E. coli 0157, but is not helpful for the detection of sorbitol-fermenting STEC other than 0157. Moreover, some strains of E. colt 0157 can ferment sorbitol. In this study, in situ hybridization using DNA probe of shiga toxin was used for the isolation of STEC from the PCR-positive stool and -Sequenbe analysis of a part of shiga toxin gene was performed. METHODS: The stool was incubated in LB broth overnight and DNA was extracted from the culture fluid. Multiplex PCR was performed with primers for stxl and stx2 genes. Specimen showed PCR-positive was incubated on MacConkey agar and colonies were blotted with nitrocellulose membrane. Digoxigenin-labelled DNA probe for shiga toxin was made by PCR and the positive colonies were detected with anti-digoxigenin-alkaline phosphatase conjugate and nitroblue tetrazolium. Agglutination test with antisera was performed for the serotying and VTEC-RPLA kit was used for the toxin production. Sequence analysis of PCR products was performed with automatic sequence analyser. RESULTS: An stxl-negative, but stx2-positive PCR was observed in a three-year-old girl, who visited Kumi Hospital on July 19, 1999 complaining of vomiting and diarrhea. The positive colonies were isolated by in situ hybridization using stx2-specific DNA probe. The titers of stxl and stx2 by VTEC-RPLA test were negative and 1:64, respectively. Agglutination for the serotyping was not observed with all of the 0 antisera. 160-nucleotide sequence of stx2 of this isolate was identical with bacteriophage 933W (GenBank X07865), except for the change (T-C) of 957th nucleotide and amino acid sequence was identical each other. CONCLUSIONS: For the sensitive detection of STEC from the stool of patients with diarrhea, multiplex PCR is recommended with stxl- and stx2-specific primers. And in situ hybridization should be performed in PCR-positive specimen for the isolation of STEC. This method may be helpful for the detection of STEC as the causative microorganisms in food-borne outbreak.

Serotyping and Phylogenetic analysis of Enteroviruses Isolated from Patients with Aspetic Meningitis / 대한임상미생물학회지

BACKGROUND: The determination of serotype of enteroviruses is useful for the discrimination between sporadic and epidemic infections. The conventional serotyping method is time-consuming and labor-intensive. Recently, molecular method was introduced for the serotyping of enteroviruses. The aim of this study was to establish a method to isolate and analyze enteroviruses from various specimens utilizing molecular biological techniques and to determine which strains were phylogenetically related to clinical samples. METHODS: Clinical samples in this study included 164 cerebrospinal fluid (CSF), 136 stool, 15 sera, 6 throat swab, 5 urine, and 4 sputa, which were obtained from hospitalized patients, primarily infants or children presenting symptoms of aseptic meningitis in 1998. RD cells were used for enterovirus isolation. RT-PCR was performed with RD cell lysate showing CPE. The primers 011 and 012 were used for the VP1 region, and the primers EN1 and EN2 for 5'-UTR. The nucleotide sequences of VP1 region were determined and analyzed with BLAST program. RESULTS: Among 333 samples, only 23 samples produced CPE: 17 samples at first and six samples at the second blind passage. Fifteen isolates were related to coxsackievirus B2 two to echovirus 4, three to echovirus 6, and three to echovirus 18. All 23 viral isolates displayed a nucleotide sequence identity of 80-95%, compared with the reference serotypes. However, the identity was increased up to 93-100% when the VP1 region was translated into amino acids CONCLUSIONS: Since CB2 type was 55% among enteroviral isolates, the CB2 was determined as the major causative serotype of enteroviral meningitis in 1998. CB2 type was emerged between June and July, EC4 and EC6 was limited to July, and EC18 was in August.

Detection of Shiga Toxin-Producing Escherichia coli by in Stitu hybridization and sequence Analysis of Stx2 / 대한임상미생물학회지

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) was found in several serotypes of E coli including 0157 serotype. Sorbitol-MacConkey agar: may be useful for the detection of E. coli 0157, but is not helpful for the detection of sorbitol-fermenting STEC other than 0157. Moreover, some strains of E. colt 0157 can ferment sorbitol. In this study, in situ hybridization using DNA probe of shiga toxin was used for the isolation of STEC from the PCR-positive stool and -Sequenbe analysis of a part of shiga toxin gene was performed. METHODS: The stool was incubated in LB broth overnight and DNA was extracted from the culture fluid. Multiplex PCR was performed with primers for stxl and stx2 genes. Specimen showed PCR-positive was incubated on MacConkey agar and colonies were blotted with nitrocellulose membrane. Digoxigenin-labelled DNA probe for shiga toxin was made by PCR and the positive colonies were detected with anti-digoxigenin-alkaline phosphatase conjugate and nitroblue tetrazolium. Agglutination test with antisera was performed for the serotying and VTEC-RPLA kit was used for the toxin production. Sequence analysis of PCR products was performed with automatic sequence analyser. RESULTS: An stxl-negative, but stx2-positive PCR was observed in a three-year-old girl, who visited Kumi Hospital on July 19, 1999 complaining of vomiting and diarrhea. The positive colonies were isolated by in situ hybridization using stx2-specific DNA probe. The titers of stxl and stx2 by VTEC-RPLA test were negative and 1:64, respectively. Agglutination for the serotyping was not observed with all of the 0 antisera. 160-nucleotide sequence of stx2 of this isolate was identical with bacteriophage 933W (GenBank X07865), except for the change (T-C) of 957th nucleotide and amino acid sequence was identical each other. CONCLUSIONS: For the sensitive detection of STEC from the stool of patients with diarrhea, multiplex PCR is recommended with stxl- and stx2-specific primers. And in situ hybridization should be performed in PCR-positive specimen for the isolation of STEC. This method may be helpful for the detection of STEC as the causative microorganisms in food-borne outbreak.

Serotyping and Phylogenetic analysis of Enteroviruses Isolated from Patients with Aspetic Meningitis / 대한임상미생물학회지

BACKGROUND: The determination of serotype of enteroviruses is useful for the discrimination between sporadic and epidemic infections. The conventional serotyping method is time-consuming and labor-intensive. Recently, molecular method was introduced for the serotyping of enteroviruses. The aim of this study was to establish a method to isolate and analyze enteroviruses from various specimens utilizing molecular biological techniques and to determine which strains were phylogenetically related to clinical samples. METHODS: Clinical samples in this study included 164 cerebrospinal fluid (CSF), 136 stool, 15 sera, 6 throat swab, 5 urine, and 4 sputa, which were obtained from hospitalized patients, primarily infants or children presenting symptoms of aseptic meningitis in 1998. RD cells were used for enterovirus isolation. RT-PCR was performed with RD cell lysate showing CPE. The primers 011 and 012 were used for the VP1 region, and the primers EN1 and EN2 for 5'-UTR. The nucleotide sequences of VP1 region were determined and analyzed with BLAST program. RESULTS: Among 333 samples, only 23 samples produced CPE: 17 samples at first and six samples at the second blind passage. Fifteen isolates were related to coxsackievirus B2 two to echovirus 4, three to echovirus 6, and three to echovirus 18. All 23 viral isolates displayed a nucleotide sequence identity of 80-95%, compared with the reference serotypes. However, the identity was increased up to 93-100% when the VP1 region was translated into amino acids CONCLUSIONS: Since CB2 type was 55% among enteroviral isolates, the CB2 was determined as the major causative serotype of enteroviral meningitis in 1998. CB2 type was emerged between June and July, EC4 and EC6 was limited to July, and EC18 was in August.

Effect of Bronchodilator and Corticosteroid Inhalation Therapy in Cough Variant Childhood Asthma / 소아알레르기및호흡기학회지

PURPOSE: Cough variant asthma may be defined as a presentation of asthma that fulfills all the criteria of asthma, inflammatory process of the airways and airway hyperresponsiveness. Because of the cough is only the manifestation in cough variant asthma especially in children, it may be very difficult to assess by the physical examination and routine spirometry. Airway function may be further evaluated by bronchial provocation, usually with methacholine or exercise, to supprot or exclude the diagnosis of asthma. In children too young to perform pulmonary function tests, a therapeutic trial can serve as a diagnostic tool. In this study we evaluate the effectiveness of inhaled bronchodilator and corticosteroid treatment to the children with cough variant asthma. METHODS: Forth-eight children who visited to our pediatric allergy clinic having a chronic cough more than 3 weeks were enrolled to our study. We defined a children who showed bronchial hyperresponsiveness after exercise challenge as a cough variant asthma. We analyzed the changes of PEFR before and after exercise and treatment for 4 weeks. RESULTS: 1) All the patients with cough variant asthma or sinobronchitis have a night aggravating cough as a sole manifestation. 2) The baseline % predicted PEFR showed within normal range in the study subjects. But there was significant decrease of % perdicted PEFR after execise challenge in the patients with cough variant asthma. 3) There was significant increase of % predicted PEFR after treatment with inhaled bronchodilator and corticosteroid in the patients with cough vatiant asthma. Also it is noted in the patients with sinobronchitis treated with antibiotics. CONCLUSION: We can find significant improvement of clinical manifestation and pulmonary function in the patients with cough variant asthma who treated with inhaled bronchodilator and corticosteoid. Because of many children suffering from chronic cough may have a cough variant asthma, we emphasized that inhaled bron-chodilator and corticosteroid treatment is effective for long-term control of cough variant asthma.