Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10-2 CCID50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 105 and 1 CCID50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 105 CCID50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses.

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.

Causality evaluation of bacterial species isolated from patients with community-acquired lower leg cellulitis.

INTRODUCTION: Lower leg cellulitis is a diffuse inflammation of the cutaneous connective tissue following invasion of microorganisms and with potential to recur. The causative agent is not routinely identified in clinical practice, and the empirical therapy initiated primarily targets the 'conventional' disease pathogens, Streptococcus pyogenes and Staphylococcus aureus. OBJECTIVE: To evaluate at case level, the role of bacterial species isolated from lesional skin in the pathogenesis of community-acquired lower leg cellulitis. METHODS: Two sampling methods (superficial swab and biopsy) were applied to isolate bacterial species from 40 patients hospitalized for first (N = 24 cases) and recurrent (N = 16 patients) lower leg cellulitis episodes. Subsequently, a clinical-laboratory heuristic algorithm was employed to interpret causality associations of isolated species with disease episodes at case level. RESULTS: In 37/40 cases (92.5%), at least one bacterial species was identified with either sampling method. The number of different species/specimen isolated from superficial swabs compared to punch biopsies was significantly more (P < 0.001). A causative agent was identified in 16 cases (40%); it was a 'conventional' pathogen in seven patients and strains belonging to one of six 'non-conventional' pathogens in nine cases. There was no concordance in the spectrum of isolated pathogens with the two sampling methods (kappa-index = 0.028). Another four species may have participated in five patients as co-pathogens in mixed infections. There was also no difference in microbiological disease features between patients with first and recurrent cellulitis episodes. CONCLUSIONS: The application of a clinical-laboratory causality algorithm coupled with pooled culture results of more than one sampling methods in patients with lower leg cellulitis is anticipated to permit the identification of responsible bacterial species at case level and offer incentive for therapeutic intervention studies.

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.

Characterization of novel intergenogroup and intergenotype recombinant noroviruses from central Greece.

Noroviruses (NoVs) are a major causative agent of acute gastroenteritis in humans. They are members of the Caliciviridae family and based on the genetic analysis of the RdRp and capsid regions, human NoVs are divided into three genogroups (Gs), GI, GII, and GIV. The three genogroups further segregate into distinct lineages called genotypes. The NoV genus is genetically diverse and recombination of viral RNA is known to depend upon various immunological and intracellular constraints that may allow the emergence of viable recombinants. In this study, three Noroviral strains detected in clinical samples revealed two hitherto unobserved recombination events between GII.9/GII.4 and GII.9/GI.7 genogroups. To our knowledge, these intergenotype and intergenogroup recombination events of GII.9/GII.4 and GII.9/GI.7, in ORF1 and ORF2 genes respectively are reported for the first time and highlight the ongoing evolution of noroviruses.

PCR assays for the identification of rare recombination types from VP1 to 3D genomic region of vaccine derived poliovirus strains.

Poliomyelitis has been effectively controlled by the use of inactivated poliovirus vaccine (IPV) or trivalent live attenuated oral poliovirus vaccine (OPV). Since 1964, the use of OPV in mass vaccinations has resulted in drastic reductions of the number of poliomyelitis cases caused by wild-type polioviruses. However, the characterization of OPV derivatives with increased neurovirulence, constituted a real problem with respect to OPV safety. Mutations at attenuating sites of the genome and recombination events between Sabin strains of the trivalent OPV vaccine have been correlated with the loss of the attenuated phenotype of OPV strains and the acquisition of traits characteristic of wild polioviruses. In consequence, early detection and characterization of recombinant evolved derivatives of vaccine strains is highly important. In this report, ten PCR assays are described which allow for the identification of rare recombination events located in VP1, 2A, 2C, 3A, 3C and 3D genomic regions and predominant recombination events located in 2C and 3D genomic regions of OPV derivatives. These assays could be readily implemented in diagnostics laboratories lacking sequencing facilities as a first approach for the early detection and characterization of recombinant OPV derivatives.

Circulation of intergenotype recombinant noroviruses GII.9/GII.6 from 2006 to 2011 in central Greece.

Noroviruses (NoVs) are members of the Caliciviridae family and are recognized as a worldwide cause of acute nonbacterial gastroenteritis. Based on the genetic analysis of the RdRp and capsid regions, human NoVs are divided into three genogroups (Gs), GI, GII, and GIV, which further segregate into distinct lineages called genotypes. In this study, in an attempt to discern the circulation of an intergenotypic recombinant GII.9/GII.6, which was previously reported by our group in central Greece, we investigated NoVs in raw sewages from 2006 to 2011 and compared the results with the viruses detected from clinical samples in the same area and in the same time period. Two specific primer pairs for NoVs were designed which amplified in a single PCR fragment from polymerase to capsid gene covering the widespread recombination point in ORF1/ORF2 junction. Based on the genetic analysis, recombinant NoV strains GII.9/GII.6 were identified. Fourteen out of 15 environmental and eight out of ten clinical samples that were used in the present study were positive, with both primer pairs, confirming that the intergenotypic recombinant GII.9/GII.6 was circulating in the population of central Greece from 2006 to 2011. The crossover point was identified to be within the overlapping region of ORF1/ORF2 (GII.9/GII.6, respectively) and was determined by Simplot at nucleotide position 5,032 bp.

Molecular and phylogenetic analysis of the HPV 16 E4 gene in cervical lesions from women in Greece.

The HPV16 E1(∧)E4 protein is thought to contribute to the release of newly formed viral particles from infected epithelia. In order to investigate amino acid mutations in the HPV16 E1(∧)E4 protein, the complete E4 ORF was amplified by PCR in 27 HPV16-positive cervical samples, and the amplicons were cloned. Fifteen nucleic acid variations were identified in the E4 ORF, including seven silent nucleic acid mutations. In addition, nine amino acid mutations (A7V, A7P, L16I, D45E, L59I, L59T, Q66P, S72F, H75Q) were detected in the E1(∧)E4 protein, and these were associated with the severity of cervical malignancy. A maximum-likelihood phylogenetic tree was constructed based on the E4 ORF, and nucleotide sequence analysis of the E4, E6 and E7 genes from the same samples was conducted in order to determine the phylogenetic origin of the cloned sequences from the amplified HPV16 E4. Based on the nucleotide sequence and phylogenetic analysis it was revealed that even though E4 ORF constitutes a small polymorphic portion of the viral genome (288 bp), it could provide valuable information about the origins of the HPV16 genome. In addition, molecular evolutionary analysis of the E4 coding region revealed that neutral selection is dominant in the overlapping region of the E4 and E2 ORFs.

Molecular characterization of a new intergenotype Norovirus GII recombinant.

Human noroviruses (NoVs) of the Caliciviridae family are a major cause of epidemic gastroenteritis. The NoV genus is genetically diverse and recombination of viral RNA is known to depend upon various immunological and intracellular constraints that may allow the emergence of viable recombinants. In the present study, we report the development of a broadly reactive RT-PCR assay, which allowed the characterization of strain A6 at molecular level, established its genetic relationship at the sub-genogroup level and classified A6 strain at the sub-genotype level. The detection was carried out initially by enzyme-linked immunosorbent assay (ELISA) and the subsequent detection and molecular characterization of NoV strain was achieved by reverse transcription-PCR and sequencing. Based on the sequence analysis, A6 strain was revealed to belong to the GII genogroup of NoVs. Partial ORF1 gene sequencing analysis and complete ORF2 gene sequencing revealed that ORF1 and ORF2 belonged to two distinct genotypes GII/9 and GII/6, respectively, making obvious that A6 strain is a rare intergenotypic recombinant within the genogroup GII between GII.9 and GII.6 genotypes. A6 strain represents the first human NoV from Greece, whose genome has been partially (ORF1&ORF3) and completed (ORF2) sequenced. To our knowledge the recombination event GII.9/GII.6 in RdRp and capsid gene, respectively, that was revealed in the present study is reported for the first time.

Retrospective molecular and phenotypic analysis of poliovirus vaccine strains isolated in Greece.

The live oral poliovirus vaccine (OPV) strains are genetically unstable, causing, in rare cases, vaccine-associated paralytic poliomyelitis. Reversions of the known attenuating mutations in OPV strains and intertypic recombination have been identified as the underlying causes of the increased neurovirulence of poliovirus isolates. In this study, three OPV isolates (one non-recombinant and two recombinants) were tested in order to correlate phenotypic traits such as temperature sensitivity (Rct test) and growth kinetics (one-step growth curve test) with mutations and recombination events of the viral genome. Moreover, the immunity level of the western Greek population aged 1-40 years was evaluated against OPV isolates and Sabin vaccine strains, with a microneutralization assay. Members of the 1-40-year age group (both pooled and individual sera) showed no significant differences in neutralization test (NT) titres against OPV isolates in comparison with the Sabin vaccine strains. However, all three OPV isolates showed reverted phenotypic traits in Rct or one-step growth curve assays. The results of our study revealed a significant decrease in immunity level from the 1-10-year age group to the 21-30-year age group (pooled sera) for both poliovirus types 1 and 3. For both poliovirus types, the highest NT titres were observed in the 1-10-year age group, and the lowest NT titre was observed in the 21-30-year age group, towards poliovirus type 3. Our study underlines the need for immunological studies in all age groups, in order to allow reconsideration of the current vaccination policies and to avoid epidemics caused by the circulation of highly evolved OPV derivatives.

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains.

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

A seroprevalence study of poliovirus antibody against a collection of recombinant and non-recombinant poliovirus vaccine strains in the population of southern Greece.

In this study, the serological status of the southern Greek population in the 1­10-year, 11­20-year, 21­30-year and 31­40-year age groups with regard to Sabin vaccine strains and a collection of 15 recombinant and four non-recombinant poliovirus vaccine strains was determined. For all three poliovirus types, the highest neutralization test (NT) titres were observed in the 1­10- year age group, indicating a good response to vaccination. In general, the serological status of the population of southern Greece with regard to poliovirus is better for types 1 and 2 than for type 3. The presence of the lowest NT titre in the 21­ 30-year age group against poliovirus type 3 suggests the need for a booster dose of monovalent Sabin3 vaccine to ensure personal and herd immunity.

A new RT-PCR assay for the identification of the predominant recombination types in 2C and 3D genomic regions of vaccine-derived poliovirus strains.

In the post-eradication era of wild polioviruses, the only remaining sources of poliovirus infection worldwide would be the vaccine-derived polioviruses (VDPVs). As the preponderance of countries certified to be polio-free has switched from OPV (oral poliovirus vaccine) to IPV (inactivated poliovirus vaccine), importation of recombinant evolved derivatives of vaccinal strains would have serious implication for public health. To test the robustness of the proposed RT-PCR screening analysis, eleven recombinant vaccine-derived polioviruses that were characterized previously by sequencing by our group, in addition to three recently identified recombinant environmental isolates were assayed. Although the most definitive characterization of VDPVs is by genomic sequencing, in this study we describe a new, inexpensive and broadly applicable RT-PCR assay for the identification of the predominant recombination types S3/Sx in 2C and S2/Sx in 3D genomic regions respectively of VDPVs, that can be readily implemented in laboratories lacking sequencing facilities as a first approach for the early detection of vaccine-derived poliovirus (VDPVs).

A theoretical investigation into post-operative, intracavitary beta therapy of high-grade glioblastomas using yttrium-90.

Beta therapy with yttrium-90 (90Y) has recently been introduced as a post-operative intra-cavitary treatment for malignant glioblastoma, a generally radioresistant tumour for which cure rates with conventional radiotherapy are usually very disappointing. This short theoretical study investigates the conditions under which 90Y treatment might be most effective and assesses the likely amounts of activity which must be infused in order to successfully cope with the low radiosensitivities which characterize such tumours. The radiobiological and physical analysis is investigated using the linear quadratic (LQ) model and a range of possible scenarios for the distribution and density of the tumour cells surrounding the surgically formed cavities are considered. The results suggest that, in the absence of diffusion of 90Y from the cavity, the activity typically required for 50% tumour cure is well over 40 mCi (1480 MBq), this being considerably more than the clinically determined activities which may be tolerated. Suggestions are provided for improving the versatility of the model.

Intramedullary spinal cord metastases in breast carcinoma: report of two cases and review of the literature.

Intramedullary spinal cord metastases (ISCM) are usually the result of rapidly progressing systemic malignancy. Breast cancer is one of the most common solid tumors with a high propensity of CNS dissemination. In the present report we describe two new cases with advanced breast cancer developing ISCM after a variable disease course. One of these patients had brain metastases at presentation, while at relapse developed leptomeningeal carcinomatosis which was treated successfully, but followed shortly, as a terminal event, by ISCM and parenchymal brain recurrence. The other patient was treated initially for locally advanced breast cancer and after multiple locoregional relapses, she developed liver metastases and subsequent ISCM and asymptomatic parenchymal brain deposits. Both patients experienced a rather rapidly evolving disease course leading to death 2 and 4 months, respectively, after widespread neuraxis dissemination of their cancer. Both these cases, added to the list of the anecdotally reported cases of ISCM after breast cancer, undermine the ominous prognosis and limited treatment options available for this disease manifestation, and an extensive literature review and discussion of similar cases is provided.

Molecular markers for the investigation of Mycobacterium gordonae epidemics.

Mycobacterium gordonae was isolated as a light growth from bronchoalveolar aspirates from nine patients over 12 months. All patients were in one hospital, and had been bronchoscoped for suspected malignancy. None of the patients had symptoms or radiographic findings of mycobacterial infection. The isolates were characterized by biochemical tests and molecular hybridization. Random amplified polymorphic DNA analysis (RAPD) was used to test whether the strains had a common origin. All the isolates generated four to eight fragments, and almost all presented distinct RAPD patterns. Antimicrobial resistance patterns to six agents confirmed that the isolates were unrelated. Thus epidemiologically unrelated strains of M. gordonae can exist as contaminants in the same department over a relatively short time frame. RAPD analysis is easy to perform, gives rapid results, and can be used for epidemiological analysis of M. gordonae isolates.

Catecholamine infusion versus intraaortic counterpulsation at the initial phase of left intraventricular balloon pumping in the fibrillating animal heart.

This experimental study compares the effect of catecholamine infusion to the effect of intraaortic counterpulsation (IABP) while initiating intraventricular balloon pumping (IVBP) in the fibrillating heart. In 12 dogs IVBP started immediately after the induction of ventricular fibrillation. Intravenous adrenaline or noradrenaline (at a progressively increasing infusion rate until the systolic aortic blood pressure was 120 mm Hg) was interchanged with IABP. The systolic aortic pressure, the aortic flow and the mean left atrial pressure were, respectively, 120.4 +/- 0.5 mm Hg, 42 +/- 4 ml kg-1 min-1 and 18.7 +/- 1.2 mm Hg (x +/- SEM) ten min after initiating catecholamine infusion and 97 +/- 5 mm Hg (with a 131 +/- 4 mm Hg diastolic wave), 69.6 +/- 4 ml kg-1 min-1 and 16 +/- 1.5 mm Hg ten min after initiating IABP. The difference in aortic flow was significant (p < 0.001). The results indicate that a better aortic flow may be obtained by combining IVBP and IABP than IVBP and vasoconstrictive agents in the fibrillating heart. If IVBP, IABP and catecholamines are combined, both AF and AP may increase.

Intraventricular plus intra-aortic balloon pumping during intractable cardiac arrest.

A method is presented for maintaining aortic flow by mechanical means during intractable cardiac arrest. A spherical balloon was inserted into the left ventricle while the usual intra-aortic balloon was introduced into the thoracic aorta. Ventricular fibrillation was induced by direct current. The pumps operating the two balloons were adjusted to inflate the intraventricular balloon during one third of the pumping cycle and the intra-aortic balloon during the next two thirds of the same cycle. The intraventricular balloon capacity varied from 40 to 110 ml (six dogs weighing 16-24 kg) while the intra-aortic balloon capacity was 20 ml. An optimal pumping rate of 75 beats/min maintained an aortic flow of 0.9-1.5 ml/beat/kg and a mean pressure into the brachiocephalic trunk of 110 +/- 12.5 mm Hg (mean +/- SD). These experimental data indicate that an easily applied mechanical device system (needing no extracorporeal circulation) may be used to bridge the time between intractable cardiac arrest and implantation of an artificial heart or transplantation.

Specific antigen and antibody to Mycobacterium leprae in the cryoprecipitate of a patient with Lucio phenomenon.

Using a sensitive and specific enzyme-linked immunosorbent assay (ELISA) assay we showed that the cryoglobulins of a patient with Lucio phenomenon contain phenolic glycolipid I antigen and a specific antibody.