Evaluation of a commercially available reverse transcription-PCR enzyme immunoassay (Enterovirus Consensus kit) for the diagnosis of enterovirus central nervous system infections

ABSTRACTA commercial reverse transcription (RT)-PCR amplification method was compared with culture for the diagnosis of enterovirus meningitis. In total, 99 cerebrospinal fluid (CSF) specimens were examined with the Enterovirus Consensus kit and shell vial culture. RT-PCR allowed the amplification of enterovirus cDNA and its detection in a microtitre plate by hybridisation. Clinical information and CSF analysis were used to resolve the discrepancy in results. The detection limit of the RT-PCR assay was determined with the Third European Union Concerted Action Enterovirus Proficiency Panel. There were 34 truepositive CSF specimens. Of these, RT-PCR detected 33 (sensitivity 97%), while culture detected 19 (sensitivity 54.5%). RT-PCR failed to detect one culture-positive specimen that contained inhibitors. When samples from the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, the RT-PCR method gave identical results to those expected. The Enterovirus Consensus kit was rapid and statistically more sensitive than culture (p < 0.01) for the detection of enteroviruses in CSF, and may offer considerable benefits in the clinical management of patients with enterovirus meningitis

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

An eternal microbe: Brucella DNA load persists for years after clinical cure

Background. Despite the continuing high incidence of brucellosis, vague aspects of pathophysiology, diagnosis, and treatment continue to exist, particularly with regard to the ability of Brucella species to survive inside the host. Methods. A quantitative real-time polymerase chain reaction assay was used for monitoring bacterial DNA load in brucellosis-affected patients throughout different disease stages. Three or more specimens per patient were obtained (1 at diagnosis, 1 at the end of treatment, and at least 1 during the follow-up period) from 39 patients with acute brucellosis. Results. The majority of patients (87% at the end of treatment, 77% at 6 months after treatment completion, and 70% at &gt;2 years after treatment) exhibited persistent detectable microbiological load despite being asymptomatic. The 3 patients who experienced relapse did not exhibit any statistically significant difference in their bacterial load at any stage of disease or during follow-up. Conclusion. Brucella melitensis DNA persists despite appropriate treatment and apparent recovery. This finding offers a new insight into the pathophysiology of the disease: B. melitensis is a noneradicable, persisting pathogen. © 2008 by the Infectious Diseases Society of America. All rights reserved

WarmStart colorimetric LAMP for the specific and rapid detection of HPV16 and HPV18 DNA

Background and objectives: Persistent infection with High-Risk HPV genotypes is the principal cause for the development of cervical cancer with HPV16 and HPV18 to be the most frequently identified HPV genotypes observed in approximately 70% of cervical cancer cases worldwide. The present study focused on the development of a simple molecular methodology based on WarmStart colorimetric LAMP for the specific identification of HPV16 and HPV18. Methods: The method was developed by designing LAMP type-specific primer sets that target the E6 gene. The assay was applied using HPV-positive clinical samples along with control cases in order to evaluate the specificity of the newly designed isothermal protocol. In addition, an experimental cutoff value was estimated through reconstitution experiments with HPV-DNA plasmids. LAMP amplicons were visualized by color changes, thus eliminating the requirement for post-amplification processing steps. Results: The WarmStart colorimetric LAMP facilitates the isothermal amplification of 10 copies per reaction of both HPV16 and HPV18 DNA, while it exhibits 100% specificity for the detection of the corresponding genotypes in LSIL and HSIL cases. Moreover, the assay demonstrates 100% PPV and 100% NPV. Finally, the sensitivity of conventional PCR with the type-specific LAMP primer sets (B3/F3)for the HPV16, HPV18 DNA detection was 100 copies/reaction and 10 copies/reaction, respectively. Conclusions: The newly established WarmStart colorimetric LAMP can be considered as a powerful molecular tool that it can be easily implemented in small clinical and research laboratories for a rapid and efficient identification of the most tumorigenic HPV genotypes. © 2019 Elsevier B.V

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

Viral agents of acute gastroenteritis in hospitalized children in Greece

A 6-year study of stool samples from 4604 children hospitalized for acute gastroenteritis was conducted to investigate the role of enteric viruses as a cause of gastroenteritis in north-west Greece. Rotaviruses, noroviruses, adenoviruses and astroviruses were detected in 21.35%, 4%, 3.5% and 2.35%, respectively, by enzyme immunoassays and molecular techniques. Molecular techniques enhanced overall diagnostic efficacy by 2.5%, and by c. 10% each for rotavirus and adenovirus. Rotavirus was the leading cause of viral gastroenteritis, usually associated with severe illness. Mixed infections were found in 4.4% of positive specimens, and rotavirus plus astrovirus represented the most frequent co-infection (55.5%). This first study on the epidemiology of viral gastroenteritis in Greece shows that recent advances in the diagnosis of viral enteropathogens may have only marginal effects on overall diagnostic efficacy, and thus the impact of viral agents causing sporadic gastroenteritis in public health cannot be fully evaluated. © Journal compilation © 2009 The Authors 2009 European Society of Clinical Microbiology and Infectious Diseases

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

Polymorphic variability in the exon 19 of the RB1 gene and its flanking intronic sequences in HPV16-associated precancerous lesions in the Greek population

Purpose. The tumour suppressor protein RB plays a decisive role in negative control of the cell cycle, inhibiting tumour development. The present analysis investigated the prevalence of the nucleotide polymorphism A153104G, which is located at intron 18 of the RB1 gene, and investigated the impact of the polymorphic variability in the exon 19 and its flanking intronic sequences on the severity of cervical disease in HPV16-positive Greek women. Methodology. The nucleotide polymorphism A153104G was detected by PCR-RFLP assay, while the amplicons were further subjected to cloning and sequencing. Moreover, molecular evolutionary analysis was performed using the maximumlikelihood (ML) and empirical Bayesian (EB) methods in order to evaluate the selective pressure acting on exon 19 of the RB1 gene. Results/Key findings. The A153104G nucleotide polymorphism was only detected in one control case. Moreover, sequence analysis of the amplicons revealed that the polymorphic variability in the RB1 gene increased with the severity of the cervical dysplasia. The link between the observed polymorphic variability and the progress of cervical disease was reflected in the molecular evolutionary analysis that was performed on the exon 19 of the RB1 gene, since negative selective pressure was acting upon exon 19 in the control and low-grade squamous intraepithelial lesion (LSIL) cervical samples, while positive selective pressure was acting upon exon 19 in the high-grade squamous intraepithelial lesion (HSIL) specimens. Conclusions. The A153104G nucleotide polymorphism did not emerge as a potential biomarker for the development of precancerous lesions in the Greek patients, while the accumulation of sequence variations in RB1 gene might influence patients’ susceptibility towards the progression of cervical neoplasia. © 2018 The Authors

Polymorphic variability in the exon 19 of the RB1 gene and its flanking intronic sequences in HPV16-associated precancerous lesions in the Greek population

Purpose. The tumour suppressor protein RB plays a decisive role in negative control of the cell cycle, inhibiting tumour development. The present analysis investigated the prevalence of the nucleotide polymorphism A153104G, which is located at intron 18 of the RB1 gene, and investigated the impact of the polymorphic variability in the exon 19 and its flanking intronic sequences on the severity of cervical disease in HPV16-positive Greek women. Methodology. The nucleotide polymorphism A153104G was detected by PCR-RFLP assay, while the amplicons were further subjected to cloning and sequencing. Moreover, molecular evolutionary analysis was performed using the maximumlikelihood (ML) and empirical Bayesian (EB) methods in order to evaluate the selective pressure acting on exon 19 of the RB1 gene. Results/Key findings. The A153104G nucleotide polymorphism was only detected in one control case. Moreover, sequence analysis of the amplicons revealed that the polymorphic variability in the RB1 gene increased with the severity of the cervical dysplasia. The link between the observed polymorphic variability and the progress of cervical disease was reflected in the molecular evolutionary analysis that was performed on the exon 19 of the RB1 gene, since negative selective pressure was acting upon exon 19 in the control and low-grade squamous intraepithelial lesion (LSIL) cervical samples, while positive selective pressure was acting upon exon 19 in the high-grade squamous intraepithelial lesion (HSIL) specimens. Conclusions. The A153104G nucleotide polymorphism did not emerge as a potential biomarker for the development of precancerous lesions in the Greek patients, while the accumulation of sequence variations in RB1 gene might influence patients’ susceptibility towards the progression of cervical neoplasia. © 2018 The Authors