- Email: [email protected]
PVII-20 A two well multiplex PCR for the diagnosis of viruses associated with childhood gastroenteritis
S42
Posters, 12th ESCV, Istanbul / Journal of Clinical Virology 46 Suppl. 1 (2009) S15–S61
Conclusions: Our results showed that the rotavirus is the frequent pathogen in acute gastroenteritis in children especially under the age two. Adenovirus infection was less common comparing to rotavirus. Detection of both viruses may be useful for epidemiological purposes. In addtion, since the rotavirus infections might have serious complications, its detection could be useful for indivudual patient.
PVII-18 Clinical efficacy of rotavirus vaccine in Turkish infants U. Tugcu1 , F. Sahin1 , G. Bozdayi2 *, F.N. Aksakal3 , G. Alp2 , S. Rota2 , U. Beyazova1 . 1 Department of Pediatrics, School of Medicine, Gazi University, Ankara, Turkey, 2 Department of Clinical Microbiology, School of Medicine, Gazi University, Ankara, Turkey, 3 Department of Public Health, School of Medicine, Gazi University, Ankara, Turkey The aim of this study is to determine the clinical efficacy of rotavirus vaccine in Turkish infants who attended to well child clinics of Gazi University Hospital between 01.05.2008 and 30.04.2009. The infants were randomly divided into two groups and 299 infants in group one received three doses of Rotateq® vaccine at 2nd, 4th and 6th months while the remaining 251 infants who did not receive any Rotavirus vaccine served as control group. The infants were followed for one year to observe if they had any diarrhea episode In groups 1 and 2, 32 (10.7%) and 33 (13.1%) infants had diarrhea respectively and the difference was insignificant (p = 0.451). Stool samples were collected from 65 infants having diarrhea during the follow up. Six (9.2%) stool samples were positive for rotavirus by ELISA (Rotaclone, Meridian Bioscience Inc., Cincinnati, OH, USA), of which 3 (9.4%) from group one and 3 (9.1%) from group two (p = 1.000). Genomic dsRNA was extracted from stool samples by phenol: chloroform: isoamylalcohol method. Electropherotyping was done by polyacrylamide gel electrophoresis (PAGE) and G typing by RT-PCR. A total of 6 samples subjected to PAGE revealed three different electropherotypes. We found three positive out of 6 samples by PCR. The G types were as follows: 1/3 was of G1 type, 1/3 G4, 1/3 G9. The P types were as follows 2/3 P[8] type, 1/3 P untypable. There was no statistically diffrerence for diarrhea between vacinated and unvaccinated children against rota virus. PVII-19 Rotavirus, rotavirus vaccine and aspect of doctors in Turkey M. Altindis1 *, R. Kalayci1 , K. Caliskan1 , R. Koken2 , K. Banyai3 . 1 Afyon Kocatepe University, School of Medicine, Dept Of Microbiology, Afyon, Turkey, 2 Afyon Kocatepe University School of Medicine, Department of Pediatry, Afyonkarahisar, Turkey, 3 Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary The aim was to investigate aspects of doctors about rotavirus in Middle Anatolia, Turkey. Totally 260 doctors (126 pediatrists, 134 family doctor and general practitioner) were included in the study. Doctors were from different cities (Afyon, Ankara, Konya, Mersin, Antalya, Kayseri, Nevsehir, Isparta, Usak, Eregli, Osmaniye) and hospitals (University, Government hospital and primary care) in Middle Anatolia. Data was collected by a questionnaire form. Most of the doctors thought about rotavirus age as 0−2 age year old groups (243/93.5%). 53.8% of the doctors thought rotavirus as the only cause in diarrhea (n:140), 75% of the doctors thought it as the cause of diarrhea plus vomiting(n:195), and plus fewer(80%, n:210), only vomiting and fewer(9%, n:23). A part of the doctors thought that rotavirus is a causative agents of nosocomial diarrhea(67%; n:175). Just a few doctors requested the fecal rotavirus antigen test when children attended clinics with diarrhae. (31.9%; n:83). Most of the doctors thought rotavirus in patients who come with the diarrhea in winter and fall seasons (80%; n:210). Most of the doctors did not know that there are two vaccines in Turkey(69.2%; n:180). Most of the doctors would advice the rotavirus vaccine(81.5%; n:212) but part of them know the rotavirus vaccines prices(60%; n:156). All of the participants thought that the rotavirus vaccines price was high. 15% of doctors applied the rotavirus vaccines in their offices(n:39). As a results the most important problem about rotavirus and its vaccines is that the virus and its vaccine is not well known among doctors and rotavirus vaccines are expensive.
PVII-20 A two well multiplex PCR for the diagnosis of viruses associated with childhood gastroenteritis D.C. Lewis1 *, M. Altindis2 , R. Koken3 , R. Kalayci2 , Y. Yoldas4 , A.D. Hale1 . Old Medical School, Leeds General Infirmary, Leeds Teaching Hospitals NHS Trust, Leeds, UK, 2 Afyon Kocatepe University, School of Medicine, Department of Microbiology, Afyonkarahisar, Turkey, 3 Afyon Kocatepe University, School of Medicine, Department of Pediatrics, Afyonkarahisar, Turkey, 4 Zubeyde Hanim Maternity Hospital and Childcare Center, Afyonkarahisar, Turkey 1 Virology,
Background: Group A rotaviruses remain the most important cause of viral gastroenteritis in children worldwide and can be diagnosed using simple inexpensive tests. However, other viruses are increasingly recognised as causing significant morbidity in this age group. Electron microscopy (EM) offers a “catch all” diagnostic method but is insensitive. We describe a two well, real time PCR that detects genetically diverse viruses associated with viral gastroenteritis. Methods: A multiplex, one step, reverse transcriptase-PCR method was designed to detect Noroviruses, group A Rotaviruses, Sapoviruses, Astroviruses, Adenoviruses and an internal control. These assays were previously published or designed in-house. The test was validated using samples submitted to teaching hospitals in the UK and Turkey for viral investigation of sporadic gastroenteritis in children (aged 16 years or less). Electron microscopy was performed on all samples negative for viral nucleic acid. Results: Two hundred faecal samples were tested in the multiplex PCR submitted during the winter season in the UK and 144 samples from symptomatic children in Turkey. All samples were negative for routinely tested bacterial pathogens. Overall, 54% of samples were positive for at least one viral pathogen with Norovirus being the commonest pathogen in both countries. EM revealed the presence of rotaviruses in 2 additional samples which were identified as Group C strains by PCR. Both samples were from Turkish children. PVII-21 Genotype specific PCR for norovirus reveals GgII.4 strains circulating in the 2008/2009 winter outbreaks in Amsterdam, the Netherlands S. Bruisten1,2 *, S. Salamat1 , J. Hubbard1 , N. Nassir1 , R. Brunst1 . 1 Public Health Laboratory (PHL), Department of Infectious Diseases, Amsterdam, the Netherlands, 2 Academic Medical Center, Laboratory of Experimental Virology, University of Amsterdam, Amsterdam, the Netherlands Gasteroenteritis caused by norovirus (NV) is well recognised nowadays. In the PHL in Amsterdam a nested PCR targeting the orf1/pol region was available since 2001. This PCR does not discriminate different genotypes, however Genogroups (Gg) II and IV can be readily detected, but not GgI. We developed a duplex real-time PCR specific for GgI and GgII targeting the orf2/caps region. In winter 2008/2009 we collected 300 stool samples and tested them for norovirus by nested PCR. Patients were associated with outbreaks of gastroenteritis (n = 41) in elderly homes, hospitals, daycare centres but we included also sporadic cases (n = 35). All samples were subsequently tested by the real-time duplex PCR. Discordant samples were retested by Genogroup specific (either GgI or GgII) real time PCR to determine an ‘end result’. A representative sample of PCR products was sequenced for genotyping and phylogeny. By nested PCR 166/300 (55.3%) samples were positive. The real-time duplex PCR was positive in 171/300 (57.0%), with GgII in 164/171 and GgI in 15 samples. However, 8/15 samples seemed to have a GgI+GgII double infection. Discrepancy analysis of 55 samples showed that none of the 15 GgI samples could be confirmed and the 8 ‘double’ positive infections were GgII positive. The sensitivity and the NPV were 100%, the specificity was 93% and the PPV 94% for the real-time duplex PCR. Sequencing analysis revealed two discriminate GgII.4 strains. We conclude that in the winter 2008/2009 season no norovirus outbreaks were missed by using the nested PCR.
Posters, 12th ESCV, Istanbul / Journal of Clinical Virology 46 Suppl. 1 (2009) S15–S61
Conclusions: Our results showed that the rotavirus is the frequent pathogen in acute gastroenteritis in children especially under the age two. Adenovirus infection was less common comparing to rotavirus. Detection of both viruses may be useful for epidemiological purposes. In addtion, since the rotavirus infections might have serious complications, its detection could be useful for indivudual patient.
PVII-18 Clinical efficacy of rotavirus vaccine in Turkish infants U. Tugcu1 , F. Sahin1 , G. Bozdayi2 *, F.N. Aksakal3 , G. Alp2 , S. Rota2 , U. Beyazova1 . 1 Department of Pediatrics, School of Medicine, Gazi University, Ankara, Turkey, 2 Department of Clinical Microbiology, School of Medicine, Gazi University, Ankara, Turkey, 3 Department of Public Health, School of Medicine, Gazi University, Ankara, Turkey The aim of this study is to determine the clinical efficacy of rotavirus vaccine in Turkish infants who attended to well child clinics of Gazi University Hospital between 01.05.2008 and 30.04.2009. The infants were randomly divided into two groups and 299 infants in group one received three doses of Rotateq® vaccine at 2nd, 4th and 6th months while the remaining 251 infants who did not receive any Rotavirus vaccine served as control group. The infants were followed for one year to observe if they had any diarrhea episode In groups 1 and 2, 32 (10.7%) and 33 (13.1%) infants had diarrhea respectively and the difference was insignificant (p = 0.451). Stool samples were collected from 65 infants having diarrhea during the follow up. Six (9.2%) stool samples were positive for rotavirus by ELISA (Rotaclone, Meridian Bioscience Inc., Cincinnati, OH, USA), of which 3 (9.4%) from group one and 3 (9.1%) from group two (p = 1.000). Genomic dsRNA was extracted from stool samples by phenol: chloroform: isoamylalcohol method. Electropherotyping was done by polyacrylamide gel electrophoresis (PAGE) and G typing by RT-PCR. A total of 6 samples subjected to PAGE revealed three different electropherotypes. We found three positive out of 6 samples by PCR. The G types were as follows: 1/3 was of G1 type, 1/3 G4, 1/3 G9. The P types were as follows 2/3 P[8] type, 1/3 P untypable. There was no statistically diffrerence for diarrhea between vacinated and unvaccinated children against rota virus. PVII-19 Rotavirus, rotavirus vaccine and aspect of doctors in Turkey M. Altindis1 *, R. Kalayci1 , K. Caliskan1 , R. Koken2 , K. Banyai3 . 1 Afyon Kocatepe University, School of Medicine, Dept Of Microbiology, Afyon, Turkey, 2 Afyon Kocatepe University School of Medicine, Department of Pediatry, Afyonkarahisar, Turkey, 3 Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary The aim was to investigate aspects of doctors about rotavirus in Middle Anatolia, Turkey. Totally 260 doctors (126 pediatrists, 134 family doctor and general practitioner) were included in the study. Doctors were from different cities (Afyon, Ankara, Konya, Mersin, Antalya, Kayseri, Nevsehir, Isparta, Usak, Eregli, Osmaniye) and hospitals (University, Government hospital and primary care) in Middle Anatolia. Data was collected by a questionnaire form. Most of the doctors thought about rotavirus age as 0−2 age year old groups (243/93.5%). 53.8% of the doctors thought rotavirus as the only cause in diarrhea (n:140), 75% of the doctors thought it as the cause of diarrhea plus vomiting(n:195), and plus fewer(80%, n:210), only vomiting and fewer(9%, n:23). A part of the doctors thought that rotavirus is a causative agents of nosocomial diarrhea(67%; n:175). Just a few doctors requested the fecal rotavirus antigen test when children attended clinics with diarrhae. (31.9%; n:83). Most of the doctors thought rotavirus in patients who come with the diarrhea in winter and fall seasons (80%; n:210). Most of the doctors did not know that there are two vaccines in Turkey(69.2%; n:180). Most of the doctors would advice the rotavirus vaccine(81.5%; n:212) but part of them know the rotavirus vaccines prices(60%; n:156). All of the participants thought that the rotavirus vaccines price was high. 15% of doctors applied the rotavirus vaccines in their offices(n:39). As a results the most important problem about rotavirus and its vaccines is that the virus and its vaccine is not well known among doctors and rotavirus vaccines are expensive.
PVII-20 A two well multiplex PCR for the diagnosis of viruses associated with childhood gastroenteritis D.C. Lewis1 *, M. Altindis2 , R. Koken3 , R. Kalayci2 , Y. Yoldas4 , A.D. Hale1 . Old Medical School, Leeds General Infirmary, Leeds Teaching Hospitals NHS Trust, Leeds, UK, 2 Afyon Kocatepe University, School of Medicine, Department of Microbiology, Afyonkarahisar, Turkey, 3 Afyon Kocatepe University, School of Medicine, Department of Pediatrics, Afyonkarahisar, Turkey, 4 Zubeyde Hanim Maternity Hospital and Childcare Center, Afyonkarahisar, Turkey 1 Virology,
Background: Group A rotaviruses remain the most important cause of viral gastroenteritis in children worldwide and can be diagnosed using simple inexpensive tests. However, other viruses are increasingly recognised as causing significant morbidity in this age group. Electron microscopy (EM) offers a “catch all” diagnostic method but is insensitive. We describe a two well, real time PCR that detects genetically diverse viruses associated with viral gastroenteritis. Methods: A multiplex, one step, reverse transcriptase-PCR method was designed to detect Noroviruses, group A Rotaviruses, Sapoviruses, Astroviruses, Adenoviruses and an internal control. These assays were previously published or designed in-house. The test was validated using samples submitted to teaching hospitals in the UK and Turkey for viral investigation of sporadic gastroenteritis in children (aged 16 years or less). Electron microscopy was performed on all samples negative for viral nucleic acid. Results: Two hundred faecal samples were tested in the multiplex PCR submitted during the winter season in the UK and 144 samples from symptomatic children in Turkey. All samples were negative for routinely tested bacterial pathogens. Overall, 54% of samples were positive for at least one viral pathogen with Norovirus being the commonest pathogen in both countries. EM revealed the presence of rotaviruses in 2 additional samples which were identified as Group C strains by PCR. Both samples were from Turkish children. PVII-21 Genotype specific PCR for norovirus reveals GgII.4 strains circulating in the 2008/2009 winter outbreaks in Amsterdam, the Netherlands S. Bruisten1,2 *, S. Salamat1 , J. Hubbard1 , N. Nassir1 , R. Brunst1 . 1 Public Health Laboratory (PHL), Department of Infectious Diseases, Amsterdam, the Netherlands, 2 Academic Medical Center, Laboratory of Experimental Virology, University of Amsterdam, Amsterdam, the Netherlands Gasteroenteritis caused by norovirus (NV) is well recognised nowadays. In the PHL in Amsterdam a nested PCR targeting the orf1/pol region was available since 2001. This PCR does not discriminate different genotypes, however Genogroups (Gg) II and IV can be readily detected, but not GgI. We developed a duplex real-time PCR specific for GgI and GgII targeting the orf2/caps region. In winter 2008/2009 we collected 300 stool samples and tested them for norovirus by nested PCR. Patients were associated with outbreaks of gastroenteritis (n = 41) in elderly homes, hospitals, daycare centres but we included also sporadic cases (n = 35). All samples were subsequently tested by the real-time duplex PCR. Discordant samples were retested by Genogroup specific (either GgI or GgII) real time PCR to determine an ‘end result’. A representative sample of PCR products was sequenced for genotyping and phylogeny. By nested PCR 166/300 (55.3%) samples were positive. The real-time duplex PCR was positive in 171/300 (57.0%), with GgII in 164/171 and GgI in 15 samples. However, 8/15 samples seemed to have a GgI+GgII double infection. Discrepancy analysis of 55 samples showed that none of the 15 GgI samples could be confirmed and the 8 ‘double’ positive infections were GgII positive. The sensitivity and the NPV were 100%, the specificity was 93% and the PPV 94% for the real-time duplex PCR. Sequencing analysis revealed two discriminate GgII.4 strains. We conclude that in the winter 2008/2009 season no norovirus outbreaks were missed by using the nested PCR.