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Abstract Background Human papillomavirus (HPV) variants differ in their biological and chemical properties, and therefore, may present differences in pathogenicity. Most authors classified variants based on the phylogenetic analysis of L1 region. Nevertheless, recombination in HPV samples is becoming a usual finding and thus, characterizing genetic variability in other regions should be essential. Objectives We aimed to characterize the genetic variability of HPV 18 in 5 genomic regions: E6, E7, E4, L1 and the Upstream Regulatory Region (URR), working with both single infection and multiple HPV infection samples. Furthermore, we aimed to assess the prevalence of HPV 18 variants in our region and look for possible existence of recombination as well as analyze the relationship between these variants and the type of lesion. Methods From 2007 to 2010, Clinical Microbiology and Infection Control Department analyzed 44 samples which were positive for HPV 18. Genetic variability was determined in PCR products and variants were assigned to European, Asian-amerindian or African lineage. Recombination and association of variants with different types of lesion was studied. Results Genetic analysis of the regions revealed a total of 56 nucleotide variations. European, African and Asian-amerindian variants were found in 25/44 (56.8%), 10/44 (22.7%) and 5/44 (11.4%) samples, respectively. We detected the presence of recombinant variants in 2/44 (4.5%) cases. Samples taken from high-grade squamous intraepithelial lesions (H-SIL) only presented variants with specific-african substitutions. Conclusions Multiple HPV infection, non-european HPV variants prevalence and existence of recombination are considered risk factors for HPV persistence and progression of intraepithelial abnormalities, and therefore, should be taken into consideration in order to help to design and optimize diagnostics protocols as well as improve epidemiologic studies. Our study is one of the few studies in Spain which analyses the genetic variability of HPV18 and we showed the importance of characterizing more than one genomic region in order to detect recombination and classify HPV variants properly.

Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of β-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for β-lactamase resistance genes. Multilocus sequence typing and β-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant β-lactamase resistance genes in less than 1 h. Multilocus sequence typing and β-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.

Community-acquired pneumonia represents the third-highest cause of mortality in industrialized countries and the first due to infection. Although guidelines for the approach to this infection model are widely implemented in international health schemes, information continually emerges that generates controversy or requires updating its management. This paper reviews the most important issues in the approach to this process, such as an aetiologic update using new molecular platforms or imaging techniques, including the diagnostic stewardship in different clinical settings. It also reviews both the Intensive Care Unit admission criteria and those of clinical stability to discharge. An update in antibiotic, in oxygen, or steroidal therapy is presented. It also analyzes the management out-of-hospital in CAP requiring hospitalization, the main factors for readmission, and an approach to therapeutic failure or rescue. Finally, the main strategies for prevention and vaccination in both immunocompetent and immunocompromised hosts are reviewed.

Background: Fast diagnosis of bloodstream infections remains the most important challenge for clinical microbiologists. The introduction of the mass-spectrometry represents a breakthrough, although several methods are already commonly used for the direct identification from positive blood cultures we present a faster method (ultra fast) for Lytic anaerobic flasks. Methods: We compare the ultra-fast (UF) method with the extensively employed differential centrifugation method (DC) and both to routine identification after 18-24 h of incubation. UF and DC method correlation rates to the gold standard were calculated, and statistical significance was proved with the Z test. Results: UF performed better overall than DC, with this difference being statistically significant. This tendency was observed in every subanalysis.

Objective: To study the prevalence and distribution of HBV genotypes in Spain for the period 2000-2016., Methods: Retrospective study recruiting 2559 patients from 17 hospitals. Distribution of HBV genotypes, as well as sex, age, geographical origin, mode of transmission, HDV-, HIV- and/or HCV-coinfection, and treatment were recorded., Results: 1924 chronically HBV native Spanish patients have been recruited. Median age was 54 years (IQR: 41-62), 69.6% male, 6.3% HIV-coinfected, 3.1% were HCV-coinfected, 1.7% HDV-co/superinfected. Genotype distribution was: 55.9% D, 33.5% A, 5.6% F, 0.8% G, and 1.9% other genotypes (E, B, H and C). HBV genotype A was closely associated with male sex, sexual transmission, and HIV-coinfection. In contrast, HBV genotype D was associated with female sex and vertical transmission. Different patterns of genotype distribution and diversity were found between different geographical regions. In addition, HBV epidemiological patterns are evolving in Spain, mainly because of immigration. Finally, similar overall rates of treatment success across all HBV genotypes were found., Conclusions: We present here the most recent data on molecular epidemiology of HBV in Spain (GEHEP010 Study). This study confirms that the HBV genotype distribution in Spain varies based on age, sex, origin, HIV-coinfection, geographical regions and epidemiological groups., Competing Interests: Declaration of Competing Interest No Competing financial and non-financial interests are disclosed by any author, (Copyright © 2020. Published by Elsevier Ltd.)

Background: Human papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, however to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease. Nowadays, mRNA expression of viral E6 and E7 HPV oncogenes stands up as a potential biomarker to improve cervical screening. We aimed to validate a method for RNA extraction, detect HPV mRNA expression and, assess the relationship between E6/E7 mRNA expression and pathology of patients' lesions and progression., Methods: This study included 50 specimens that had been previously genotyped as HPV16, 18, 31, 33 and/or 45. Cervical swabs were extracted with three different RNA extraction methods -Nuclisens manual extraction kit (bioMérieux), High Pure Viral RNA Kit (Roche) and RNeasy Plus Mini kit (Qiagen)-, and mRNA was detected with NucliSens EasyQ HPV version 1 test (bioMérieux) afterwards. Association of oncogene expression with pathology and lesion progression was analyzed for each extraction method., Results: E6/E7 mRNA positivity rate was higher in samples analyzed with bioMérieux (62%), followed by Roche (24%) and Qiagen (6%). Women with lesions and lesion progression showed a higher prevalence of viral RNA expression than women that had not lesions or with lesion persistence. While bioMérieux revealed a higher sensitivity (77.27%), Roche presented a higher PPV (75%) and an increased specificity (89.28%)., Conclusions: Extraction methods based on magnetic beads provided better RNA yield than those based in columns. Both Nuclisens manual extraction kit (bioMérieux) and High Pure Viral RNA Kit (Roche) seemed to be adequate for E6/E7 mRNA detection. However, none of them revealed both high sensitivity and specificity values. Further studies are needed to obtain and validate a standard gold method for RNA expression detection, to be included as part of the routine cervical screening program.

Background: The aims of this study were to detect HPV E6/E7 mRNA expression in women with high-risk genotypes (HPV-16, -18, -31, -33 and -45) analysing its relationship with tissue pathology and 2) 2-year follow-up of E6/E7 mRNA tested group., Methods: Our samples were genotyped and classified by pathologists according to Bethesda system. After RNA extraction, E6/E7 oncogene mRNA detection was performed by NucliSens® EasyQ® HPV v1 Test (bioMérieux)., Results: The results of the present study showed that E6/E7 mRNA positivity rate was 68.29 % in women tested once and 69.56 % in women tested twice. According to tissue pathology, all samples with high-grade lesions were positive for mRNA. Among women with low-grade lesions varied over the years from 89.28 to 84 % in women tested once and from 77.77 to 70 % in tested twice. Among women without lesion, positivity rate maintained in women tested once (from 50 to 41.38 %) and decreased in tested twice, from 63.63 to 44.44 %. Regarding lesion evolution, mRNA positivity was higher in women with lesion progression (53.13 %) and in women with positive results in two tested samples (83.33 %)., Conclusion: HPV E6/E7 mRNA detection may be an effective screening test and biomarker for cervical cancer in women infected with these five genotypes. Nonetheless, further studies are needed to standardize as routine triage test.

Background: In the literature, data on the prevalence of human papillomavirus (HPV) infection in men vary significantly and the exact distribution of specific genotypes is still unclear. As infections usually occur without symptoms, men might only attend their hospital clinic when they have a specific concern, being in most cases genital warts (condylomas), which are often caused by low-risk HPV genotypes. The aim of this study was to assess HPV genotype distribution and prevalence among men attending hospital for HPV-associated conditions and to evaluate infection-associated factors., Methods: Samples from men with clinical manifestations of HPV-related infections seen during 2007-2012 at the Clinical Microbiology and Infectious Control Department at Basurto University Hospital were genotyped using Linear Array HPV Genotyping Test kit (Roche Molecular Diagnostics, Germany). Data on probable risk factors were collected and investigated for possible association., Results: Of 184 anogenital samples, 138 (75 %) were tested as positive for HPV; 57 (41.3 %) single HPV infections and 81 (58.7 %) multiple infections. Only 45.6 % of HPV-positive samples presented low-risk genotypes 6 or 11, whereas 71/138 (51.4 %) had at least one oncogenic (high-risk) genotype. Oncogenic genotypes and multiple HPV infections were both associated with a higher number of lifetime sexual partners and their incidence appeared to increase with patient age., Conclusions: Although it is accepted that HPV 6 and 11 genotypes are main causes of condylomas, our findings show a high incidence of multiple infections and high-risk genotypes in men with benign HPV manifestations. The fact that the condyloma is a skin lesion facilitates the entry of virus into cells and thus cancer progression; therefore, monitoring for HPV is important, especially in those patients with high-risk genotypes (regardless of whether they cause condyloma).

Aims: The aims of the study were (1) to characterize the genetic variability of human papillomavirus (HPV) genotype 16 in the E6 region when this genotype is present in multiple infection samples, (2) to assess the prevalence of variants in our region and (3) to analyze the relationship between variants, patients' ages and pathology., Methods: The Clinical Microbiology and Infection Control Department analyzed samples which were positive for genotype 16 and other genotypes from 2007 to 2013. Variants were assigned to European, Euro-German, Asian, Asian-American or African lineage by sequence analysis. The relationship among variants, age and different types of lesion was studied., Results: In HPV-16 sequence analysis, the European variant was detected in 85.10% of samples, the Asian-American in 7.80%, the African in 4.25% and the Euro-German in 2.83% of specimens. Sequence genetic variability showed 16 nucleotide substitutions. Moreover, non-European variants were mainly found in old women and in isolates from high-grade squamous intraepithelial lesions since European variants were mainly detected in negative cytologies., Conclusion: Multiple infections may take effect on nucleotide substitution and the appearance of recombinant samples. Single gene analysis makes it impossible to detect recombination which has a great influence on drug response and vaccine strategies. Thus, E6 gene analysis would be enough to identify HPV-16 intratypic variants but not to confirm the results.

Objective: Hepatitis B virus (HBV) genotyping and detection of resistance to drugs have become essential in epidemiological and clinical diagnosis. Our main objective was to determine the prevalence of HBV genotypes and drug-resistance mutations in chronic asymptomatic carriers and chronic HBV sufferers comparing 2 detection assays., Methods: Serum samples from 28 chronic HBV patients and 22 chronic asymptomatic carriers were analyzed. For HBV genotyping, the INNO-LIPA and TRUGENE™ HBV genotyping kits were evaluated. For drug-resistance mutations, INNO-LIPA DR v2 and INNO-LIPA DR v3 prototype and the TRUGENE™ HBV genotyping kit were evaluated., Results: In HBV genotyping, concordant results were 98% and both assays were able to detect more than one genotype. Different genotypes were detected, the most prevalent being D (46%) and A (26%). In relation to drug-resistance mutations, the sensitivity of the line probe assay was lower than TRUGENE because INNO-LIPA could not detect two mutations (S202G and V214A)., Conclusions: Both assays are easy and suitable for detecting HBV genotype and drug-resistance mutations and for routine laboratory use. However, TRUGENE presented better sensitivity in both analyses and it is possible to conduct both on the same sample. This assay is also able to detect primary and secondary mutations., (Copyright © 2013 S. Karger AG, Basel.)

Background: Long-term lamivudine (LAM), adefovir (ADV) and entecavir (ETV) treatment induce the emergence of drug-resistant hepatitis B virus (HBV) in patients with chronic hepatitis B infection., Aim: To evaluate the LAM, ADV and ETV resistance mutations detected in our patient group., Materials and Methods: Twenty patients who had received at least two years of treatment with nucleoside/tide analogues were enrolled in this study. Patients with detectable HBV DNA were analyzed in order to detect resistance mutations and in this group of patients treatment was change., Results: Three patients developed LAM resistance mutations (2 presented rtM204I and one rtL180M+rtM204V/I) and one patient showed rtN236T ADV resistance mutation. During ADV and LAM treatment, one patient developed ADV plus LAM resistance mutations (rtI163V+rtL180M+rtA181V+rtN236T), in this case, HBV strains harbouring polymerase mutations did not develop LAM associated rtM204V/I primary mutation. In addition, ETV resistance mutations (rtL180M+rtT184A+rtS202G+rtM204V) were detected in one patient., Conclusions: These findings suggest that monotherapy resulted in a limited virological response and combination strategies including potent antiviral agents should be recommended for patients with resistant mutations.

Genomic mutations occurring during reverse transcription of hepatitis B virus (HBV) could explain its genetic diversity and account for 8 genetically distinct genotypes that are geographically distributed quite differently. The main objectives of this study were to determine the prevalence of hepatitis B virus genotypes in patients with chronic hepatitis B and to see if there was a relationship between genotypes and risk factors for transmission based on HBeAg status. A total of 14 serum samples were analyzed using INNO-LIPA HBV genotyping assay. Genotype D was the most prevalent (64.3%) followed by genotype A (28.6%). There was one case of co-infection (D/E genotypes) that was confirmed by PCR sequencing. All patients except one were HBeAg-negative and anti-HBe-positive. The risk factors for HBV transmission were unknown in half of the cases; in the other half, sexual, transfusion, maternal or interfamilial transmission were observed. The results show that genotype D is the most prevalent genotype in our hospital, followed by genotype A. On the other hand, no relationship was found between HBeAg status and genotype.

Background: To establish the rate of HGV/GB virus C (GBV-C) transmission by blood components in open-heart surgery patients., Study Design and Methods: From 55 patients receiving blood components, sera were collected before and 2, 4, 6, 8, 10, 12, 16, 20, 26, and 32 weeks after heart surgery. Serum samples from patients and implicated blood donations were tested for HGV/GBV-C RNA by PCR. Recipients of RNA-positive blood components were also tested for the presence of E2 antibodies (E2Ab) by ELISA., Results: Of 55 recipients, 18 received RNA-positive blood components. Of 14 recipients of RNA-positive blood components, who were negative for RNA or E2Ab before transfusion, 8 became RNA positive and one developed E2Ab after transfusion. Three recipients of RNA-positive blood components had E2Ab before transfusion, and none of these became RNA positive after transfusion. One of 18 recipients was RNA positive before and after transfusion. Of 55 recipients, 37 received RNA-negative blood components: 34 were RNA negative before and after transfusion. Of 37 recipients, 3 were RNA positive before and after transfusion., Conclusion: Of susceptible patients, 64 percent became infected with HGV/GVC-C when transfused with RNA-positive blood components. E2Ab-positive patients were protected against HGV/GBV-C infection.

The most important aim of this study was to describe the hypothetical relationship between the PePHD region variability (related to the synthesis of a cellular enzyme pseudosubstrate) of the hepatitis C virus and the response of patients to interferon therapy. This interaction could be a determining factor in the antiviral effect of interferon. All samples (from 24 patients with chronic hepatitis C infection) were analyzed using a previously described method based on RT-PCR and nested PCR mediated by single-strand conformation polymorphism assay (SSCP). The patients were divided into three groups with respect to the response to therapy: 8 patients with sustained response, 8 patients with transient response and 8 nonresponders. In all samples a low genetic heterogeneity pattern was detected, which was independent of other factors involved in the lack of response to treatment, such as age, sex or viral genotype. This genetic homogeneity is an indirect indication of the importance of the region on viral persistence. However, more studies are needed to evaluate the real role of this sequence on the interaction between cells and the virus.

The aim of this study was to evaluate the relationship between the quasispecies in the HVR1 region of the hepatitis C virus and treatment evolution in order to determine whether genetic complexity is predictive of response to interferon therapy. The samples were analyzed by nested RT-PCR-mediated single-strand conformation polymorphism assay (SSCP). Twelve patients with chronic hepatitis C were studied and divided into three groups: three patients with sustained response, three patients with transient response and six nonresponders. The patients in the sustained response group showed a low genetic complexity pattern. By contrast, in three nonresponders and in one patient with transient response, the SSCP assay revealed a high complexity pattern. With regard to the remaining patients with transient response, new SSCP bands appeared, thereby modifying their genetic complexity pattern. Therefore, nonresponse to interferon treatment could be related to the presence of a high genetic complexity pattern, while the detection of a low genetic complexity pattern is necessary for a positive response to interferon therapy. Due to the limited number of patients involved in this study, it was not possible to predict the response to interferon based on the genetic complexity pattern. Larger studies are therefore required.

Comparison of 33 epidemiologically distinct GBV-C/hepatitis G virus complete genome sequences suggests the existence of four major phylogenetic groupings that are equally divergent from the chimpanzee isolate GBV-C(tro) and have distinct geographical distributions. These four groupings are not consistently reproduced by analysis of the virus 5'-noncoding region (5'-NCR), or of individual genes or subgenomic fragments with the exception of the E2 gene as a whole or of 200-600 nucleotide fragments from its 3' half. This region is upstream of a proposed anti-sense reading frame and contains conserved potential RNA secondary structures that may be capable of directing the internal initiation of translation. Phylogenetic analysis of this region from certain South African isolates is consistent with previous analysis of the 5'-NCR suggesting that these belong to a fifth group. The geographical distribution of virus variants is consistent with a long evolutionary history that may parallel that of pre-historic human migrations, implying that the long-term evolution of this RNA virus is extremely slow.

Enterococci are a frequent cause of nosocomial and community infections, especially the E. faecalis and E. faecium species. They often show intrinsic resistance to cephalosporins and quinolones, and acquired resistance to other antimicrobials, such as glycopeptides, has also been described. In order to test the impact of antibiotic resistance in enterococci isolated from infections, we carried out a multicenter study in 19 hospitals in Spain. We verified whether resistance to a high concentration of aminopenicillins and aminoglycosides was high (30.86% for ampicillin, 32.32% for gentamicin at a 500 micrograms dose, 55.93% for streptomycin at a 1000 micrograms dose) while the resistance to glycopeptides was low (1.8% for vancomycin and 1% for teicoplanin). It was also shown that teicoplanin had greater intrinsic activity than vancomycin, with teicoplanin 0.5 mg/l inhibiting 86.1% of the strains studied, whereas only 12.8% were inhibited with the same concentration of vancomycin.

Objective: We aimed to investigate the modifications of HCV RNA (genomic and antigenomic strands) in peripheral blood mononuclear cells (PBMCs) of long-term responder patients to alpha-interferon therapy, and their usefulness as criteria of definitive HCV eradication., Methods: We studied 10 patients with chronic hepatitis C with > 1 yr of sustained response after alpha-interferon therapy (normal alanine aminotransferase [ALT] and negative serum HCV RNA). Serum HCV RNA and genotyping were determined. Approximately 2 and 4 yr after completion of treatment we investigated the presence of HCV RNA (genomic and antigenomic strands) in PBMCs. Eight of 10 patients were rebiopsed 2 yr after discontinuation of treatment., Results: The mean follow-up was 46.6 +/- 4.6 months (range, 39-51 months). In this period, all patients remained in sustained response. In the first determination, all patients had HCV RNA genomic strands and two patients had antigenomic strands detectable in PBMCs. Two years later only two patients had genomic and none had antigenomic strands detectable. After 4 yr of sustained response, eight of 10 patients lost HCV RNA from PBMCs., Conclusions: In the long-term follow-up, the majority of patients with chronic hepatitis C with sustained response after alpha-interferon therapy progressively lost HCV RNA from PBMCs. This determination in PBMCs is not a predictor of response.

The seroprevalence of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) markers was evaluated in a group of 2042 subjects from a rural area in the Republic of Equatorial Guinea, to obtain a better understanding of the transmission patterns of these viruses. Antibodies to HIV-1 were detected in 12 subjects (0.6%); the seroprevalence did not differ significantly by age or gender. Overall seroprevalence for HCV was 1.7% (in patients aged > 40 years, 5.6%). Hepatitis B surface antigen was detected in 8.8% of subjects, with the higher seroprevalence in children aged < or = 18 years of 13.4% contrasting with the higher seroprevalence of HCV in older subjects of the Equatoguinean studied population. These results indicate differences in the distribution of the viruses and, probably, different routes of transmission. The study demonstrates the existence of a high HBV carrier rate in children, concluding that hepatitis B vaccine should be incorporated into the Expanded Programme on Vaccination in Equatorial Guinea.

Background: The aim of this study was to develop molecular biology techniques as hybridization with oligonucleotide probes to characterize TEM-1 and TEM-2 beta-lactamases in strains of Salmonella spp., Methods: Twenty seven strains of Salmonella spp. were selected. Twenty six were resistant to ampicillin due to the production of beta-lactamases enzymes of pl of 5.4 and/or 5.6 corresponding to TEM-type. Initially, they were submitted to colony hybridization with a 420 bp. TEM probe obtained from plasmid pBR322. The strains with positive signal were selected to perform colony hybridization and Southern blot with oligonucleotide probes for TEM-1 (Gln 37) and TEM-2 (Lys 37). Finally, polymerase chain reaction technique (PCR) was developed to obtain DNA., Results: Only 17 out of the 26 beta-lactamase producing strains gave positive signals with the TEM intragenic probe. Experiments with the oligonucleotide probes in colony hybridization did not allow us discriminate positive from negative signals. Southern blot of DNA obtained from alkaline lysates did not work as we could not obtain any signals in the filters. To resolve these problems and to obtain enough DNA to perform Southern blot we developed PCR. This way it was able discriminate the bla-TEM-1 from the bla-TEM-2 genes., Conclusions: PCR technique plus oligonucleotide probes are a good alternative for the specific characterization of TEM-1 and TEM-2 beta-lactamases in Salmonella spp.

Sheep plasma alpha 1-protease inhibitor (PI1) variants were typed by analysing neuraminidase treated samples by isoelectric focusing in ultrathin polyacrylamide gels followed by staining for trypsin inhibition. The 10 different PI1 phenotypes observed were shown to be controlled by four codominant alleles. The probability of sire exclusion provided by PI1 polymorphism was about 0.45 in both of the studied milk sheep breeds (Latxa and Karrantzar) of the Basque Country.

Background: The purpose of this study was to develop a satisfactory technique to detect punctual mutations in blaTEM genes., Methods: The strains [E. coli HB 101 pBR322 (TEM-1), E. coli J62 RP4 (TEM-2)] were submitted to PCR with primers PL1 and PL2 which amplify the genetic region susceptible of punctual mutations. Then, we developed Southern blot and hybridization with oligonucleotide probes (GIn 37, Lys 37 y Thr 261), corresponding to first and last mutations., Results: A region of 841 bp was amplified using the primers previously described. Hybridization experiments with the Thr 261 probe gave positive signal with both strains (both carry the mutation); GIn 37 only hybridized with TEM-1 and Lys 37 only with TEM-2., Conclusions: The use of primers which amplify all the region susceptible of mutations in blaTEM and oligonucleotide probes allows the specific detection of point modifications in the original genes by the use of digoxigenin-labeled oligonucleotide probes.

Background: The presence of hepatitis delta virus was investigated in liver biopsies by in situ cyto-hybridization with a probe labelled with digoxigenin., Methods: The techniques developed included extraction of plasmid DNA by lysis by alkali, electroelution, electrophoresis in agarose gel and digoxigenin labelling, and the application in liver tissue., Results: Viral RNA was detected in 6 of the 10 patients, and the reactivity was exclusively restricted to the nucleolus of the hepatocytes., Conclusion: This method reveals as a sensitive and quick diagnostic procedure, which allows to study the intensity of the infection as well as the serological state of the patient.