Judit Deák

Comparison of an ELISA and two reverse transcription polymerase chain reaction methods for norovirus detection

Noroviruses are uncultivable; ELISA and reverse transcription polymerase chain reaction (RT-PCR) methods are therefore widely used for their detection. Sixty-one sporadic, diarrhoeal stool samples from various university hospital wards and from outpatients in Szeged, Hungary, were examined. Three methods were compared: two RT-PCR methods (the Argene Calici/Astrovirus Consensus kit and the Cepheid Norovirus Primer and Probe Set) and one ELISA method (the IDEIA™ Norovirus ELISA Test). Sensitivities of 78.9%, 92.8%, and 91.2%, and specificities of 100%, 100%, and 100% were found for the IDEIA™ Norovirus ELISA, the Argene kit, and the Cepheid kit, respectively. The PCR and ELISA systems detected 52 norovirus-positive samples, one of which belonged to genogroup I and all the others to genogroup II. Although the ELISA kit has a lower sensitivity compared to the PCR ones, it can be useful for large-scale testing. However, ELISA-negative outbreaks should be retested by RT-PCR methods. Our results suggest that noroviruses, and predominantly genogroup II of the norovirus genera, play an important role in outbreaks and sporadic cases of acute gastroenteritis, not only in infants and young children, but also in adults.

Large-scale whole genome sequencing identifies country-wide spread of an emerging G9P[8] rotavirus strain in Hungary, 2012

With the availability of rotavirus vaccines routine strain surveillance has been launched or continued in many countries worldwide. In this study relevant information is provided from Hungary in order to extend knowledge about circulating rotavirus strains. Direct sequencing of the RT-PCR products obtained by VP7 and VP4 genes specific primer sets was utilized as routine laboratory method. In addition we explored the advantage of random primed RT-PCR and semiconductor sequencing of the whole genome of selected strains. During the study year, 2012, we identified an increase in the prevalence of G9P[8] strains across the country. This genotype combination predominated in seven out of nine study sites (detection rates, 45-83%). In addition to G9P[8]s, epidemiologically major strains included genotypes G1P[8] (34.2%), G2P[4] (13.5%), and G4P[8] (7.4%), whereas unusual and rare strains were G3P[8] (1%), G2P[8] (0.5%), G1P[4] (0.2%), G3P[4] (0.2%), and G3P[9] (0.2%). Whole genome analysis of 125 Hungarian human rotaviruses identified nine major genotype constellations and uncovered both intra- and intergenogroup reassortment events in circulating strains. Intergenogroup reassortment resulted in several unusual genotype constellations, including mono-reassortant G1P[8] and G9P[8] strains whose genotype 1 (Wa-like) backbone gene constellations contained DS1-like NSP2 and VP3 genes, respectively, as well as, a putative bovine-feline G3P[9] reassortant strain. The conserved genomic constellations of epidemiologically major genotypes suggested the clonal spread of the re-emerging G9P[8] genotype and several co-circulating strains (e.g., G1P[8] and G2P[4]) in many study sites during 2012. Of interest, medically important G2P[4] strains carried bovine-like VP1 and VP6 genes in their genotype constellation. No evidence for vaccine associated selection, or, interaction between wild-type and vaccine strains was obtained. In conclusion, this study reports the reemergence of G9P[8] strains across the country and indicates the robustness of whole genome sequencing in routine rotavirus strain surveillance.

Surveillance of human rotaviruses in 2007–2011, Hungary: Exploring the genetic relatedness between vaccine and field strains

BACKGROUND The availability of rotavirus vaccines has resulted in an intensification of post vaccine strain surveillance efforts worldwide to gain information on the impact of vaccines on prevalence of circulating rotavirus strains. OBJECTIVES In this study, the distribution of human rotavirus G and P types in Hungary is reported. In addition, the VP4 and VP7 genes of G1P[8] strains were sequenced to monitor if vaccine-derived strains were introduced and/or some strains/lineages were selected against. STUDY DESIGN The study was conducted in 8 geographic areas of Hungary between 2007 and 2011. Rotavirus positive stool samples were collected from diarrheic patients mostly <5 years of age. Viral RNA was amplified by multiplex genotyping RT-PCR assay, targeting the medically most important G and P types. When needed, sequencing of the VP7 and VP4 genes was performed. RESULTS In total, 2380 strains were genotyped. During the 5-year surveillance we observed the dominating prevalence of genotype G1P[8] (44.87%) strains, followed by G4P[8] (23.4%), G2P[4] (14.75%) and G9P[8] (6.81%) genotypes. Uncommon strains were identified in a low percentage of samples (4.12%). Phylogenetic analysis of 318 G1P[8] strains identified 55 strains similar to the Rotarix strain (nt sequence identities; VP7, up to 97.9%; VP4, up to 98.5%) although their vaccine origin was unlikely. CONCLUSIONS Current vaccines would have protected against the majority of identified rotavirus genotypes. A better understanding of the potential long-term effect of vaccine use on epidemiology and evolutionary dynamics of co-circulating wild type strains requires continuous strain surveillance.

First Detection of P[6],G9 Rotaviruses in Hungary—An Imported Strain From India?

EuroRotaNet was launched to monitor rotavirus strain prevalence during and after introduction of rotavirus vaccines in Europe. In early 2007, we detected P[6],G9 rotaviruses to appear in Hungary, representing the first documented occurrence of this strain in our surveillance area. Epidemiologic data suggested that this strain was introduced from India.

Screening for Chlamydia trachomatis in asymptomatic women in Hungary

Background. A multicenter survey was carried out in order to determine the prevalence and risk factors for Chlamydia trachomatis infection in the population of asymptomatic women in Hungary. Results were used to carry out a cost‐effectiveness analysis of screening for chlamydial infection in women with asymptomatic genital infections.

Application of DNA Chip Scanning Technology for Automatic Detection of Chlamydia trachomatis and Chlamydia pneumoniae Inclusions

ABSTRACT Chlamydiae are obligate intracellular bacteria that propagate in the inclusion, a specific niche inside the host cell. The standard method for counting chlamydiae is immunofluorescent staining and manual counting of chlamydial inclusions. High- or medium-throughput estimation of the reduction in chlamydial inclusions should be the basis of testing antichlamydial compounds and other drugs that positively or negatively influence chlamydial growth, yet low-throughput manual counting is the common approach. To overcome the time-consuming and subjective manual counting, we developed an automatic inclusion-counting system based on a commercially available DNA chip scanner. Fluorescently labeled inclusions are detected by the scanner, and the image is processed by ChlamyCount, a custom plug-in of the ImageJ software environment. ChlamyCount was able to measure the inclusion counts over a 1-log-unit dynamic range with a high correlation to the theoretical counts. ChlamyCount was capable of accurately determining the MICs of the novel antimicrobial compound PCC00213 and the already known antichlamydial antibiotics moxifloxacin and tetracycline. ChlamyCount was also able to measure the chlamydial growth-altering effect of drugs that influence host-bacterium interaction, such as gamma interferon, DEAE-dextran, and cycloheximide. ChlamyCount is an easily adaptable system for testing antichlamydial antimicrobials and other compounds that influence Chlamydia-host interactions.

Transcriptome Analysis Indicates an Enhanced Activation of Adaptive and Innate Immunity by Chlamydia-Infected Murine Epithelial Cells Treated with Interferon γ

BACKGROUND Interferon γ (IFN‐γ) is the major cytokine involved in the elimination of Chlamydia infection. Despite its importance, the combined effect of Chlamydia infection and IFN‐γ on the gene expression of murine epithelial cells has only partially been described. METHODS The DNA chip method was used to evaluate the impact of IFN‐γ and both the human strain Chlamydia trachomatis L2 infection and the murine strain Chlamydia muridarum infection on the transcriptome of murine epithelial cells. RESULTS The gene expression analysis revealed that IFN‐γ had an enhancing effect on both the up‐regulation and down‐regulation of the epithelial gene expression. The influenced gene functional classes included cytokine and chemokine expression, antigen presentation, apoptosis, and genes involved in basic metabolic processes such as fatty acid oxidation. We also detected the up‐regulation of various genes that could be directly antichlamydial, such as members of the p47 GTPase family, inducible nitric oxide synthase, and monokine induced by IFN‐γ (MIG). As a functional validation of DNA chip data, we measured the antichlamydial effect of MIG on the extracellular form of Chlamydia. CONCLUSIONS Our results show that IFN‐γ is a key cytokine that primes epithelial cells to activate adaptive and innate immunity and to express antichlamydial effector genes both intracellularly and extracellularly.

Post vaccination rotavirus surveillance in Hungary, in 2007

Vaccination is the main strategy to control severe dehydrating gastroenteritis caused by rotaviruses in early childhood. The availability of new generation rotavirus vaccines has led to an intensification of strain surveillance worldwide, in part, to gauge the impact of the possible vaccine-driven immune selection of wild-type rotavirus strains. In the present study, authors describe the strain prevalence data obtained in 2007, with the involvement of different regions of Hungary. Genomic RNA was extracted from rotavirus-positive stool samples collected mainly from children and then subjected to genotyping using multiplex RT-PCR assay. Type-specific primers targeted G1 to G4, G6, G8 to G10, and G12 VP7 specificities, and P[4], P[6], and P[8] to P[11] VP4 specificities were used. Out of 489 rotavirus-positive specimens, collected from 482 patients, 466 and 474 were successfully G and P typed, respectively, and both G and P type specificities could be assigned for 457 strains. Prevalence data showed the predominance of G4P[8] (31.5%) strains, followed by G1P[8] (28.3%), G2P[4] (19.3%), and G9P[8] (10.2%). Minority strains were G1P[4] (0.4%), G2P[8] (1.3%), G3P[9] (0.2%), G4P[6] (0.7%), G6P[9] (0.4%), G8P[8] (0.2%), G9P[4] (0.2%), G9P[6] (0.8%), and G12P[8] (0.4%). Mixed infections were found in 1.2% of the samples, while 4.9% remained partially or fully non-typified. Our data indicate that the antigen specificities of medically important rotavirus strains identified in this 1-year study are well represented in the vaccines available in the pharmaceutical private market in Hungary. Depending on the vaccination coverage achievable in the forthcoming years, the post-vaccination rotavirus strain surveillance may allow us to gain comprehensive information on the impact of rotavirus vaccines on the prevalence of circulating rotavirus strains.

[EuroRotaNet--European rotavirus strain surveillance network established with Hungarian participation].

Group A rotaviruses are the most common cause of severe gastroenteritis worldwide. The incidence and distribution of group A rotavirus sero/genotypes varies between geographical areas during a rotavirus season, and from one season to the next. In addition, cocirculation of genetically diverse multitypic rotaviruses and of intratypic variants in any one place and time is common. Assuming widespread use of rotavirus vaccine in the near future, comprehensive surveillance of natural rotavirus infections is vital. EuroRotaNet has been established in order to gather comprehensive information on the rotavirus types co-circulating throughout Europe. The main objectives of the network are to (i) develop methods and algorithms for effective rotavirus strain typing and characterisation, (ii) describe in detail the molecular epidemiology of rotavirus infections in Europe, (iii) monitor the effectiveness of current genotyping methods and respond to changes associated with genetic drift and shift, and (iv) monitor the emergence and spread of novel rotavirus strains within Europe. This infrastructure may serve as a platform for future surveillance activities and nested studies for evaluating the effectiveness of a rotavirus vaccine in the general population. Studies to monitor the reduction in disease associated with common rotavirus types, the possible vaccine-induced emergence of antibody escape mutants of genotypes other than those included in the vaccine and of reassortment between vaccine and naturally circulating wildtype strains are required.Az A-csoportu rotavirusok vilagszerte a gyermekkori sulyos gasztroenteritiszek leggyakoribb okai. A rotavirusok szero- es genotipusainak kulonboző foldrajzi teruleteken eszlelt megoszlasa ugyanabban a szezonban es az egymast kovető evekben egyarant valtozik. Jellemző ezenkivul a genetikailag egymastol elterő torzsek egyuttes előfordulasa, akarcsak az adott tipuson beluli variabilitas barmely teruleten es időben. A rotavirus elleni vakcinak szeles korű alkalmazasat feltetelezve a kozeljovőben a termeszetes rotavirus-fertőzesek atfogo surveillance-a alapvető fontossagu lesz. Az EuroRotaNet mindenekelőtt azert jott letre, hogy atfogo informaciot gyűjtson az Europaban keringő rotavirusok szero- es genotipusairol. A megfogalmazott feladatok kozul a legfontosabbak a kovetkezők: (i) a rotavirustorzsek hatekony tipizalasi es jellemzesi modszerenek es algoritmusok kifejlesztese; (ii) a rotavirus-fertőzesek molekularis epidemiologiajanak reszletes leirasa Europaban; (iii) a jelenlegi genotipizalasi modszerek hateko...

Combination of selective enrichment and MALDI-TOF MS for rapid detection of Streptococcus agalactiae colonisation of pregnant women

Sample preparation was optimized for MALDI-TOF MS directly from selective enrichment broth to detect Streptococcus agalactiae. The method was tested on 100 vaginal samples of pregnant women; positive and negative predictive values were 100 and 91%, respectively. If it indicates positivity, colonisation can be reported 18-24h after sample collection.