Molecular approaches for HPV genotyping and HPV-DNA physical status

Persistent infection with high-risk human papillomavirus (HPV) genotypes is the leading cause of cervical cancer development. To this end several studies have focused on designing molecular assays for HPV genotyping, which are considered as the gold standard for the early diagnosis of HPV infection. Moreover, the tendency of HPV DNA to be integrated into the host chromosome is a determining event for cervical oncogenesis. Thus, the establishment of molecular techniques was promoted in order to investigate the physical status of the HPV DNA and the locus of viral insertion into the host chromosome. The molecular approaches that have been developed recently facilitate the collection of a wide spectrum of valuable information specific to each individual patient and therefore can significantly contribute to the establishment of a personalised prognosis, diagnosis and treatment of HPV-positive patients. The present review focuses on state of the art molecular assays for HPV detection and genotyping for intra-lesion analyses, it examines molecular approaches for the determination of HPV-DNA physical status and it discusses the criteria for selecting the most appropriate regions of viral DNA to be incorporated in HPV genotyping and in the determination of HPV-DNA physical status. Copyright © Cambridge University Press 2017

Enteroviral infection in Greek AIDS patients

Objective: Prolonged intestinal replication of polioviruses has not previously been studied in Greek AIDS patients. The objective of our study was to estimate the prevalence of enteroviral infections in this population. Methods: Nineteen stool samples were investigated from 19 different patients. Collection took place at the Hellenic Red Cross Hospital, Athens, Greece, between August and October 2002. Samples were processed as follows: virus isolation was attempted by cell culture using three different cell lines (human epidermoid carcinoma [Hep]-2, rabdomyosarcoma [RD], and mouse cells genetically modified in order to express the polio virus receptor in their cell surface [L20B]). An enterovirus-specific reverse transcription (RT)-PCR was then applied. Finally, seroneutralization tests were performed on 11 blood samples taken from a number of the patients who had supplied stool samples. Results: Samples were negative for enterovirus detection of any serotype on all cell lines. No cytopathic effect was observed. Enterovirus-specific RT-PCR assays were also negative for the detection of enteroviral RNA. Seroneutralization revealed relatively high antibody titers against poliovirus 1 and 2 in three of the eleven blood samples. Conclusions: Greek AIDS patients are not vulnerable to enteroviral infections and do not constitute a potential reservoir of poliovirus-prolonged excretion in Greece

Environmental surveillance of circulation of enteroviruses-Public health effects

Objective: This study presents a new approach for the molecular "serotyping" of enteroviruses concentrated and isolated from urban sewage. Method: Samples were collected from the Nicosia Sewage Treatment Plant in Cyprus. Viruses were adsorbed on cellulose nitrate membrane filters, isolated from the membrane filter using the VIRADEN method and identified by RT-PCR, followed by 5-UTR RFLP analysis and partial sequencing of the VP1 protein coding region. Results: In total, 42 enteroviruses were isolated. Initial sub-grouping based on HpaII restriction profile showed that all isolates belonged to the same genetic subcluster of non-polio enteroviruses. Partial VP1 sequencing revealed that isolates belonged to serotypes CBV4 (33 isolates, 79%), CBV1 (8 isolates, 19%) and Echovirus 7 (1 isolate, 2%). All isolates of the same serotype had highly similar VP1 sequences. The HpaII digests predicted the genetic sub-cluster in all cases. Finally, phylogenetic analysis revealed that the isolates correlate with strains of the same serotype isolated during the last four decades in Europe, Asia, Northern Africa and the Middle East, implying a possible epidemiological relationship. Conclusions: In conclusion, the results confirm that this methodology is highly promising for environmental surveillance of enterovirus circulation and epidemiology. 5-UTR RFLP analysis with HpaII is useful for initial sub-classification, while partial VP1sequencing is efficient for molecular serotyping

Novel molecular techniques for the identification of the members of Brucellaceae family

An account is presented of an attempt to identify a multiresistant, gram-negative bacterium that was isolated from raw sewage in Cyprus. The API 2ONE system was initially used to identify the strain on the basis of its biochemical profile. In addition, a 16S rRNA-specific PCR was applied on genetic material extracted directly from BGM cell culture material after which the sequence of the largest part of the 16S rRNA gene (approximately 1000 nucleotides) was obtained BLAST software was used to compare the sequences obtained with corresponding sequences of other strains belonging to members of the family Brucellaceae, which were available in the GenBank database. The isolated bacterium was identified as Ochrobactrum anthropi by both its biochemical profile and its 16S rRNA sequences, which showed a 98% similarity with other O. anthropi strains. It was found to be resistant to several antibiotics, including b-lactams and aminoglycosides. Phylogenetic analysis showed that 16S rRNA gene sequencing should be used with caution for the identification of family Brucellaceae members that are genetically closely related. In conclusion, this study recorded the first isolation of an O. anthropi strain from environmental samples in Cyprus. This strain was found to be highly resistant to antibiotics. Sequencing of the 16S rRNA gene was useful for the verification of the identity of the strain. However, phylogenetic comparisons with other related bacterial strains showed that this method is perhaps not the most decisive for identification of members of the family Brucellaceae, due to the consonance between different species and genera

The Balkan region: NDM-1-producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece

Objectives: Despite the fact that the NDM-1 carbapenemase has successfully disseminated worldwide, outbreaks remain uncommon in the European region. We describe the characteristics of the first outbreaks caused by NDM-1-producing Klebsiella pneumoniae clonal isolates in Greece. Methods: Between January 2010 and June 2013, 132 non-repetitive carbapenem-resistant Enterobacteriaceae isolates, which gave a positive modified Hodge test and were phenotypically suspected of metallo-b-lactamase production, were recovered from patients hospitalized at Ioannina University Hospital. Resistance genes were identified by PCR and sequencing. Plasmid profiling, conjugation experiments, enterobacterial repetitive intergenic consensus PCR, PFGE and multilocus sequence typing (MLST) were performed. Patient records were retrieved to access patterns of acquisition. Results: Molecular testing verified the presence in 78 K. pneumoniae isolates, collected from 71 patients, of theblaNDM-1 gene. The blaCTX-M-15, blaOXA-1 and blaTEM-1 genes were also present in most isolates. The blaNDM-1 gene was located on a narrow host range IncFII-type plasmid, of ̃95 kb, flanked upstream by a non-truncated ISAba125 element and downstream by the bleMBL gene. Genotyping clustered all K. pneumoniae isolates into a single clonal type with one subtype and MLST assigned them to sequence type 11. Two outbreaks were noted, the first between November and December 2011 involving four patients and the second initiated in May 2012 and ongoing, involving the remaining patients. All but two cases were characterized as hospital acquired. No links to immigration or travel history to endemic areas were established. Conclusions: This survey highlights the successful undetected dissemination of yet another carbapenemase in Greece and strengthens the hypothesis of a latent NDM-1 cluster in the Balkan region. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved

Development of a multiplex RT-PCR assay for the identification of recombination types at different genomic regions of vaccine-derived polioviruses

Polioviruses (PVs) are the causal agents of acute paralytic poliomyelitis. Since the 1960s, poliomyelitis has been effectively controlled by the use of two vaccines containing all three serotypes of PVs, the inactivated poliovirus vaccine and the live attenuated oral poliovirus vaccine (OPV). Despite the success of OPV in polio eradication programme, a significant disadvantage was revealed: the emergence of vaccine-associated paralytic poliomyelitis (VAPP). VAPP is the result of accumulated mutations and putative recombination events located at the genome of attenuated vaccine Sabin strains. In the present study, ten Sabin isolates derived from OPV vaccinees and environmental samples were studied in order to identify recombination types located from VP1 to 3D genomic regions of virus genome. The experimental procedure that was followed was virus RNA extraction, reverse transcription to convert the virus genome into cDNA, PCR and multiplex-PCR using specific designed primers able to localize and identify each recombination following agarose gel electrophoresis. This multiplex RT-PCR assay allows for the immediate detection and identification of multiple recombination types located at the viral genome of OPV derivatives. After the eradication of wild PVs, the remaining sources of poliovirus infection worldwide would be the OPV derivatives. As a consequence, the immediate detection and molecular characterization of recombinant derivatives are important to avoid epidemics due to the circulation of neurovirulent viral strains. © 2016, Springer Science+Business Media New York

Complete genomic characterization of an intertypic Sabin 3/Sabin 2 capsid recombinant

The genetic properties of strain K/2002, isolated from fecal samples of a 7-month-old child who had received his first oral poliovirus vaccine (OPV) dose at the age of 3 months, are described. Preliminary sequencing characterization of isolate K/2002 revealed an S3/S2 recombination event at the 3' end of the VP1 coding region. A recombination event resulted in the introduction of six Sabin 2 amino acid residues in a Sabin 3 genomic background. Furthermore, mutations associated with loss of the attenuated phenotype of Sabin 3 strains have been identified in the genome of isolate K/2002. The data presented here emphasize the need for careful planning of vaccination strategies, which involve stopping OPV administration in regions that are certified to be polio-free

Direct extraction and molecular characterization of enteroviruses genomes from human faecal samples

Routine diagnosis of acute flaccid paralysis (AFP) is still based on classical virological procedures. Several enteroviruses serotypes are not easily isolated in cell cultures system used and routinely more than one passage in cell culture is performed. A total of 54 archived faecal samples were examined. The heterogeneous nature of faecal samples may contribute to variations in the yields of viral nucleic acids with different extraction methods and specimen types. PCR inhibitors are frequently encountered in stool specimens. From the three methods initially compared for extraction of viral RNA, QIAamp Viral RNA Mini Kit was retained as it yielded the highest amount of viral RNA without the interference of RTPCR inhibitors. Evaluation of 54 archived stool specimens by RT-PCR and cell culture resulted in a higher frequency of detection by RT-PCR. With the use of RT-PCR we were able to detect two additional samples otherwise considered negative for enterovirus isolation if only the cell culture standard methodology was employed. RNA extraction with QIAamp Viral RNA Mini Kit coupled with RT-PCR in the 5'NCR (sub-grouping into distinct genetic clusters of all enteroviruses) and VP1 (reliable serotyping by sequencing) is a rapid and sensitive technique of direct poliovirus/non-polio enteroviruses recovery and molecular characterization from human faecal specimens without further passage in cell culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen. (C) 2008 Elsevier Ltd. All rights reserved

Molecular identification and full genome analysis of an echovirus 7 strain isolated from the environment in Greece

Two enteroviruses from river water and four from sewage treatment plant were isolated in Larissa, Greece, that all shared the same sequence. A full genome analysis was conducted in an attempt to reveal the evolutionary pathways of one of the isolated strains (LR11F7). VP1 nucleotide and phylogenetic analysis revealed that the isolated strain had 78% homology with the echovirus 7 prototype strain Wallace. Full genome analysis revealed that LR11F7 P1 region is related to echoviruses 7 and that P2 and P3 regions are originating from contemporary enteroviruses isolated in South Asia. Two recombination events were shown to be involved into the evolutionary history of LR11F7, the one event concerning 3A, 3B, and 2C, and the other concerning 3D genomic region, both with new types of HEV-B. The contribution of recombination to enterovirus evolution is substantial, giving rise to new genetic lineages with unknown properties

Serum Neutralization Assay for the Determination of Antibody Levels Against Non-Polio Enterovirus Strains in Central and Western Greece

Mutations and recombination events have been identified in enteroviruses. Point mutations accumulate with a frequency of 6.3 × 10-4 per base pair per replication cycle affecting the fitness, the circulation, and the infectivity of enteroviral strains. In the present report, the serological status of the Central and Western Greek population (Larissa and Ioannina, respectively) in the 1-10-year, 11-20-year, 21-30-year, and 31-40-year age groups against six non-polio enterovirus strains, their respective echovirus prototypes, and Sabin 1, 2, and 3 vaccine strains was evaluated, through serum-neutralization assay. In the Western Greek population, antibody levels were detected only for clinical isolates of E30 serotype in all age groups, and for environmental isolate LR61G3 (E6 serotype) only in the 31-40 age group, whereas an immunity level was observed in the Central Greek population, against all strains, except for EIS6B (E3 serotype). Amino acid substitutions were encountered across the structural region of the capsid, between the prototypes and the respective isolates. These substitutions may alter the antigenicity of each strain and may explain the variations observed in the neutralization titers of the different strains. As a consequence, these substitutions severely affect antibody binding and increase the ability of the virus to escape the immune response. It is tempting to assume that changes in the antigenic properties observed in circulating echoviruses represent a selection of viral variants that are less prone to be neutralized by human antibodies. These facts argue for the need of immunological studies to the population to avoid epidemics due to the circulation of highly evolved derivatives. © Copyright 2016, Mary Ann Liebert, Inc