Ultrafast detection of ß-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing.

Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of ß-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 ß-lactamase resistance genes. Multilocus sequence typing and ß-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant ß-lactamase resistance genes in less than 1 h. Multilocus sequence typing and ß-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.

Klebsiella pneumoniae is a bacterium that can cause infections in the blood. These infections can be severe, especially if K. pneumoniae is not susceptible to antibiotics ('antibiotic resistant'). Tools that can detect this resistance are important. In this study, we tested one such tool called MinION™ with blood samples. In 1 h, we were able to identify the bacteria within the sample and their resistance. This type of testing would help clinicians to give the best treatment to patients. More studies are needed to prove the usefulness of MinION for processing samples from real patients.

Cluster of cases due to Shigella flexneri producing CTX-M-15 in Spain.

The aim of our study was to delineate an outbreak of gastroenteritis caused by Shigella flexneri and affecting sixteen persons between May and June 2014 in Bilbao, Spain. All patients exhibited symptoms after consuming kebab in the same kebab shop.The outbreak is described through the clinical cases, the microbiological and molecular genetic diagnosis, and the epidemiologic investigation. Minimum inhibitory concentrations for ampicillin, amoxicillin plus clavulanic acid, third and fourth generation cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, co-trimoxazole, colistin and tigecycline were measured. The S. flexneri strains were screened by PCR for TEM, SHV, CTX-M beta-lactamases and plasmidic AmpCs and aac(6')-Ib gene. Serotyping, pulsed field gel-electrophoresis, conjugation assay, plasmid sizing by S1 enzyme digestion and Southern blot hybridization were accomplished.All the S. flexneri isolates proved to be serotype 2 and produced extended-spectrum beta-lactamase (ESBL). Carbapenems, fluoroquinolones, tigecycline, colistin, and co-trimoxazole remained active antibiotics. All the strains harboured blaCTX-M-15 and blaOXA-1 genes. The strains hosted two high-molecular weight plasmids of 100 and 230 kb, respectively. According to the hybridization assay blaCTX-M-15 was located on the plasmid of 230 kb. The identical pulsotype verified the presence of outbreak.Remarkable, that one of the food handlers has travelled recently to Pakistan, where ESBL-producing Shigella strains had been reported previously. To the best of our knowledge, this is the first outbreak caused by CTX-M-15-expressing S. flexneri in Spain and as well as in Europe.

Diagnosis of Superficial Mycoses by a Rapid and Effective PCR Method from Samples of Scales, Nails and Hair.

Superficial mycoses are the most frequently diagnosed affections of the stratum corneum of the skin, nails and hair. It is generally caused by the presence of yeasts and dermatophytes. Onychomycosis is the most common infection with an incidence of 80-90% in Europe generally produced by Trichophyton rubrum. The aim of this study is to compare the traditional diagnostic techniques of superficial mycoses with a homemade and wide-spectrum fungal polymerase chain reaction (PCR) technique that amplifies a specific region of the 18S ribosomal RNA (rRNA) directly from samples of scales, nails and hair. A total of 626 clinical samples (obtained in the Basurto University Hospital, Bilbao, Spain) were analysed by traditional culture, microscopy and PCR. DNA extraction was carried out by using an extraction buffer and bovine serum, and amplification of samples and performance of the PCR were checked by conventional agarose gel electrophoresis with subsequent sequencing of amplified samples. A total of 211 samples (34%) resulted in positive diagnosis with at least one of the two applied methods: culture (21%) and PCR (22%). Despite the low percentage of identification achieved by the sequencing technique (40%), the value contributed by the amplification of the 18S region of the rRNA was considered important in the identification as it showed a high predictive values for both positive and negative diagnoses (90.9% and 94.6%, respectively). The proposed PCR method has been confirmed as a complementary, rapid, and effective method in the diagnosis of superficial mycoses. Additionally, it reduces the time to obtain satisfactory results from 4 weeks to 7 h.

Antimicrobial susceptibility trends and evolution of isolates with extended spectrum Beta-lactamases among Gram-negative organisms recovered during the SMART study in Spain (2011-2015) / Evolución de la sensibilidad y de los aislados productores de Beta-lactamasas de espectro extendido en microorganismos gramnegativos en el estudio SMART en España (2011-2015)

Introduction. The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study monitors antimicrobial susceptibility and extended spectrum β-lactamases (ESBLs) in Gram-negative bacilli recovered from intra-abdominal infections (IAI). Material and methods. Antimicrobial susceptibility of 5,343 isolates from IAI recovered in 11 centres during the 2011-2015 SMART-Spain program was analysed by standard microdilution (EUCAST criteria) and compared with that from 2002-2010. ESBLs were phenotypically detected. Results. Escherichia coli, the most common isolate, significantly decreased in community acquired IAI (60.9% 2002-2010 vs. 56.1% 2011-2015, P=0.0003). It was followed in prevalence by Klebsiella pneumoniae that increased both in the community (8.9% vs. 10.8%, P=0.016) and nosocomial (9.2% vs. 10.8%, P=0.029) IAI and P. aeruginosa, which significantly increased in community acquired IAI (5.6% vs. 8.0%, P=0.0003). ESBLs were more prevalent in K. pneumoniae (16.3%) than in E. coli(9.5%) of nosocomial origin and were more frequently isolated from elderly patients (>60 years). Considering all Enterobacteriaceae, ertapenem (92.3-100%) and amikacin (95.5%-100%) were the most active antimicrobials. Ertapenem activity, unlike amoxicillin-clavulanate or piperacillin-tazobactam, remained virtually unchanged in ESBL (100%) and non-ESBL (98.8%) E. coli producers. Its activity decreased in ESBL-K. pneumonia (74.7%) but was higher than that of amoxicillin-clavulanate (14.0%) and piperacillin-tazobactam (24.0%). Interestingly, ertapenem susceptibility was maintained in >60% of ESBL isolates that were resistant to amoxicillin-clavulanate, piperacillin-tazobactam or luoroquinolones. Conclusions. SMART-Spain results support current guidelines which include ertapenem as empiric treatment in mild-moderate community-acquired IAI, particularly with ESBL producers. These recommendations will need to be updated with the recently introduction of new antimicrobials

Introducción. El estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) monitoriza la sensibilidad antimicrobiana y las β-lactamasas de espectro extendido (BLEE) en bacilos gramnegativos obtenidos de infecciones intraabdominales (IIA). Material y Métodos. Se ha analizado la sensibilidad antimicrobiana (microdilución estándar, criterios EUCAST) y las BLEE (detección fenotípica) de 5.343 aislados de IIA en 11 centros del programa SMART-España durante 2011-2015 en comparación con 2002-2010. Resultados. Escherichia coli, el microorganismo más prevalente, disminuyó significativamente en las IIA de origen comunitario (60,9% 2002-2010 vs. 56,1% 2011-2015, P=0,0003). Fue seguido en prevalencia por Klebsiella pneumoniae que aumentó tanto en IIA comunitaria (8,9% vs. 10,8%, P=0,016) como nosocomial (9,2% vs. 10,8%, P=0,029) y por P. aeruginosa que aumentó en la IIA comunitaria (5,6% vs. 8,0%, P=0,0003). Las BLEE fueron más prevalentes en la IIA nosocomial por K. pneumoniae(16,3%) que por E. coli(9,5%), siendo más frecuentes en pacientes de mayor edad (>60 años). Considerando todas las Enterobacteriaceae, ertapenem (92,3-100%) y amikacina (95,5%-100%) fueron los antimicrobianos más activos. La sensibilidad a ertapenem, al contrario que a amoxicilina-clavulánico o piperacilina-tazobactam, se mantuvo sin cambios en E. coli con (98,8%) y sin BLEE (100%). Su sensibilidad disminuyó en BLEE-K. pneumoniae (74,7%) pero fue mayor que la de amoxicilina-clavulánico (14,0%) o piperacilina-tazobactam (24,0%). Es de resaltar que esta actividad se mantuvo >60% en los aislados con BLEE resistentes a amoxicilina-clavulánico, piperacilina-tazobactam o fluoroquinolonas.Conclusiones. El estudio SMART-España sustenta las guías actuales que incluyen al ertapenem como tratamiento empírico en la IIA leve-moderada comunitaria, en particular con BLEE. Estas recomendaciones precisaran actualizarse con la reciente introducción de nuevos antimicrobianos

RNA extraction method is crucial for human papillomavirus E6/E7 oncogenes detection.

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.

Assessment of human papillomavirus E6/E7 oncogene expression as cervical disease biomarker.

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.

Transmission dynamics of HIV-1 subtype B in the Basque Country, Spain.

This work was aimed to study the HIV-1 subtype B epidemics in the Basque Country, Spain. 1727 HIV-1 subtype B sequences comprising protease and reverse transcriptase (PR/RT) coding regions, sampled between 2001 and 2008, were analyzed. 156 transmission clusters were detected by means of phylogenetic analyses. Most of them comprised less than 4 individuals and, in total, they included 441 patients. Six clusters comprised 10 or more patients and were further analyzed in order to study their origin and diversification. Four clusters included men who had unprotected homosexual sex (MSM), one group was formed by intravenous drug users (IDUs), and another included both IDUs and people infected through unprotected heterosexual sex (HTs). Most of these clusters originated from the mid-1980s to the mid-1990s. Only one cluster, formed by MSM, originated after 2000. The time between infections was significantly lower in MSM groups than in those containing IDUs (P-value <0.0001). Nucleoside RT and non-nucleoside RT inhibitor (NRTI and NNRTI)-resistance mutations to antiretroviral treatment were found in these six clusters except the most recent MSM group, but only the IDU clusters presented protease inhibitor (PI)-resistance mutations. The most prevalent mutations for each inhibitor class were PI L90M, NRTI T215D/Y/F, and NNRTI K103N, which were also among the most prevalent resistant variants in the whole dataset. In conclusion, while most infections occur as isolated introductions into the population, the number of infections found to be epidemiologically related within the Basque Country is significant. Public health control measures should be reinforced to prevent the further expansion of transmission clusters and resistant mutations occurring within them.

The use of Zostavax in Spain: the economic case for vaccination of individuals aged 50 years and older.

Background Population aging brings up a number of health issues, one of which is an increased incidence of herpes zoster (HZ) and its complication, post-herpetic neuralgia (PHN). Zostavax vaccine has recently become available to prevent HZ and PHN. This study evaluates the cost-effectiveness of vaccination against HZ in Spain considering a vaccination of the population aged 50 years and older and comparing this to the current situation where no vaccination is being administered. Methods An existing, validated, and published economic model was adapted to Spain using relevant local input parameters and costs from 2013. Results Vaccinating 30% of the Spanish population aged 50 years and older resulted in €16,577/QALY gained, €2025/HZ case avoided, and €5594/PHN case avoided under the third-party payer perspective. From a societal perspective, the ICERs increased by 6%, due to the higher price of the vaccine. The number needed to vaccinate to prevent one case was 20 for HZ, and 63 for PHN3. Sensitivity analyses showed that the model was most sensitive to the HZ and PHN epidemiological data, the health state utilities values, and vaccine price used. Conclusion Considering an acceptable range of cost-effectiveness of €30,000-€50,000 per QALY gained, vaccination of the 50+ population in Spain against HZ with a new vaccine, Zostavax, is cost-effective and makes good use of the valuable healthcare budget.

Distribution of genital human papillomavirus genotypes in benign clinical manifestations among men from Northern Spain.

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).

Phylogeny and Phylogeography of a Recent HIV-1 Subtype F Outbreak among Men Who Have Sex with Men in Spain Deriving from a Cluster with a Wide Geographic Circulation in Western Europe.

We recently reported the rapid expansion of an HIV-1 subtype F cluster among men who have sex with men (MSM) in the region of Galicia, Northwest Spain. Here we update this outbreak, analyze near full-length genomes, determine phylogenetic relationships, and estimate its origin. For this study, we used sequences of HIV-1 protease-reverse transcriptase and env V3 region, and for 17 samples, near full-length genome sequences were obtained. Phylogenetic analyses were performed via maximum likelihood. Locations and times of most recent common ancestors were estimated using Bayesian inference. Among samples analyzed by us, 100 HIV-1 F1 subsubtype infections of monophyletic origin were diagnosed in Spain, including 88 in Galicia and 12 in four other regions. Most viruses (n = 90) grouped in a subcluster (Galician subcluster), while 7 from Valladolid (Central Spain) grouped in another subcluster. At least 94 individuals were sexually-infected males and at least 71 were MSM. Seventeen near full-length genomes were uniformly of F1 subsubtype. Through similarity searches and phylogenetic analyses, we identified 18 viruses from four other Western European countries [Switzerland (n = 8), Belgium (n = 5), France (n = 3), and United Kingdom (n = 2)] and one from Brazil, from samples collected in 2005-2011, which branched within the subtype F cluster, outside of both Spanish subclusters, most of them corresponding to recently infected individuals. The most probable geographic origin and age of the Galician subcluster was Ferrol, Northwest Galicia, around 2007, while the Western European cluster probably emerged in Switzerland around 2002. In conclusion, a recently expanded HIV-1 subtype F cluster, the largest non-subtype B cluster reported in Western Europe, continues to spread among MSM in Spain; this cluster is part of a larger cluster with a wide geographic circulation in diverse Western European countries.

Abordaje multidisciplinar de la infección por virus del papiloma humano / Multidisciplinary approach to human papillomavirus infection

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Undertreatment of human immunodeficiency virus in psychiatric inpatients: a cross-sectional study of seroprevalence and associated factors.

BACKGROUND: The aims of this study were to evaluate the prevalence of HIV and its associated demographic and clinical factors among psychiatric inpatients of a general hospital. METHODS: This was a single-center, observational, cross-sectional study that included patients consecutively admitted to our unit aged 16 years or older and with no relevant cognitive problems. The patients were evaluated using a semistructured interview and an appropriate test for HIV infection. RESULTS: Of the 637 patients who were screened, 546 (86%) who consented to participate were included in the analyses. Twenty-five (4.6%, 95% confidence interval [CI] 3.0-6.8) patients were HIV-positive. The prevalence was higher among patients with substance misuse (17.4%, 95% CI 9.7-28.8). All except one of the 25 patients knew of their seropositive condition prior to participation in the study. Only 14 (56%) of the 25 seropositive patients had previously received pharmacological treatment for their infection. According to the multiple logistic regression analysis, the likelihood of HIV infection was lower in patients with higher levels of education and higher among patients who were single, had history of intravenous drug use, and had an HIV-positive partner, particularly if they did not use condoms. Among the patients with HIV infection, 18 (72%) had a history of suicide attempts compared with 181 (34.7%) of the patients without HIV infection (relative risk 2.1, 95% CI 1.6-2.7; P<0.001). CONCLUSION: HIV infection is highly prevalent in patients admitted to a psychiatric unit, especially those with a diagnosis of substance misuse. Seropositive patients show very poor treatment adherence. The risk of suicide seems to be very high in this population. Implementing interventions to reduce the suicide risk and improve adherence to antiretroviral therapy and psychotropic medications seems crucial.

Human Papillomavirus 16 Variants May Be Identified by E6 Gene Analysis.

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.

Comparison of INNO-LIPA and TRUGENE assays for genotyping and drug-resistance mutations in chronic hepatitis B virus infection.

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.

Human papillomavirus (HPV) genotype 18 variants in patients with clinical manifestations of HPV related infections in Bilbao, Spain.

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.

Antimicrobial susceptibility of Gram-negative organisms from intraabdominal infections and evolution of isolates with extended spectrum ß-lactamases in the SMART study in Spain (2002-2010).

INTRODUCTION: The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study records the antimicrobial susceptibility of Gram-negative bacilli obtain from intraabdominal infections with special focus in isolates with extended spectrum ß-lactamases (ESBLs). MATERIAL AND METHODS: The antimicrobial susceptibility of 8,869 isolates was analyzed by microdilution during the SMART study performed in Spain from 2002 to 2010. Isolates were recovered in 16 centres. RESULTS: Escherichia coli was the most prevalent pathogen (60.9% from intraabdominal infections acquired in the community and 49.9% in those from nosocomial origin) followed by Klebsiella pneumoniae (8.9% vs 9.2%). Pseudomonas aeruginosa was more common in intraabdominal infections from nosocomial origin (5.6% community and 8.6% nosocomial). Frequency of ESBL-producing isolates was: E. coli, 8.7%; K. pneumoniae, 8.4%; Klebsiella oxytoca, 1.4%; and Proteus mirabilis, 1.6%. Overall, ESBL-producing isolates were more frequently isolated from elderly patients (6.8% >60 years). Ertapenem and meropenem were the most active antimicrobials (susceptibility range with EUCAST criteria, 89.0-100%) when considering all Enterobacteriaceae isolates and also against ESBL producers (95.5-100%). Susceptibility of amoxicillin/clavulanic acid and piperacillin/tazobactam was lower, particularly among ESBL-producing isolates. Nevertheless, ertapenem maintained a good activity (susceptibility >95%) in ESBL-producers that were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam or fluoroquinolones. CONCLUSIONS: Antimicrobial susceptibility data from the SMART-Spain study reinforce current therapeutic guidelines of intraabdominal infections that include ertapenem as the empirical choice for treatment. This is also supported by the high frequency of ESBL-producers in our geographic area.

Antimicrobial susceptibility of Gram-negative organisms from intra abdominal infections and evolution of isolates with extended spectrum Beta-lactamases in the SMART study in Spain (2002-2010)

Introducción. El estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) tiene como objetivo monitorizar la sensibilidad a los antimicrobianos de los microorganismos gramnegativos aislados en la infección intraabdominal, con especial seguimiento de los que producen Beta-lactamasas de espectro extendido (BLEE). Material y métodos. Se han analizado por microdulución los datos de sensibilidad de 8.869 aislados recogidos en el estudio SMART en España entre 2002 y 2010 en el que han participado 16 centros. Resultados. Escherichia coli fue el patógeno más frecuente (60,9% en la infección intraabdominal adquirida en la comunidad y 49,9% en la nosocomial) seguido de Klebsiella pneumoniae (8,9% vs 9,2%). Pseudomonas aeruginosa fue más habitual en la infección nosocomial (5,6% comunitaria y 8,6% nosocomial). La frecuencia de aislados con BLEE fue: E. coli 8,7%, K. pneumoniae 8,4%, Klebsiella oxytoca 1,4% y Proteus mirabilis 1,6%. En los pacientes de mayor edad aumentó la proporción global de aislados con BLEE (6,8% en pacientes >60 años). Ertapenem y meropenem fueron los antimicrobianos más activos en el conjunto de las enterobacterias (rango de sensibilidad con criterios EUCAST, 89- 100%) y también entre los aislados con BLEE (95,5-100%). La actividad de amoxicilina/ácido clavulánico y piperacilina/tazobactam fue considerablemente inferior, en particular en los aislados con BLEE. Ertapenem mantuvo una buena actividad (sensibilidad >95%) en los productores de BLEE resistentes a amoxicilina/ácido clavulánico, piperacilina/tazobactam o fluoroquinolonas. Conclusiones. Los datos de sensibilidad del estudio SMART en España avalan las guías terapéuticas actuales de infecciónintraabdominal que sitúan al ertapenem como tratamiento empírico de elección, teniendo en cuenta sobre todo la elevada frecuencia de aislados con BLEE en nuestro medio(AU)

Introduction. The SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study records the antimicrobial susceptibility of Gram-negative bacilli obtain from intraabdominal infections with special focus in isolates with extended spectrum Beta-lactamases (ESBLs). Material and Methods. The antimicrobial susceptibility of 8,869 isolates was analyzed by microdilution during the SMART study performed in Spain from 2002 to 2010. Isolates were recovered in 16 centres. Results. Escherichia coli was the most prevalent pathogen (60.9% from intraabdominal infections acquired in the community and 49.9% in those from nosocomial origin) followed by Klebsiella pneumoniae (8.9% vs 9.2%). Pseudomonas aeruginosa was more common in intraabdominal infections from nosocomial origin (5.6% community and 8.6% nosocomial). Frequency of ESBL-producing isolates was: E. coli, 8.7%; K. pneumoniae, 8.4%; Klebsiella oxytoca, 1.4%; and Proteus mirabilis, 1.6%. Overall, ESBL-producing isolates were more frequently isolated from elderly patients (6.8% >60 years). Ertapenem and meropenem were the most active antimicrobials (susceptibility range with EUCAST criteria, 89.0-100%) when considering all Enterobacteriaceae isolates and also against ESBL producers (95.5-100%). Susceptibility of amoxicillin/clavulanic acid and piperacillin/tazobactam was lower, particularly among ESBL-producing isolates. Nevertheless, ertapenem maintained a good activity (susceptibility >95%) in ESBL-producers that were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam or fluoroquinolones. Conclusions. Antimicrobial susceptibility data from the SMART-Spain study reinforce current therapeutic guidelines of intraabdominal infections that include ertapenem as the empirical choice for treatment. This is also supported by the high frequency of ESBL-producers in our geographic area(AU)

Utilidad de la determinación del pH vaginal para el diagnóstico de las vulvovaginitis y su relación con la patología obstétrica / Utility of vaginal pH determination in the diagnosis of vulvovaginitis and its association with obstetric pathology

Objetivos. Valorar la utilidad de la determinación del pH vaginal en el primer control del embarazo para el diagnóstico de vulvovaginitis y su relación con la patología obstétrica. Material y métodos. Hemos realizado el estudio en 200 embarazadas de bajo riesgo. Efectuamos el pH vaginal en el primer control del embarazo a las gestantes. Independientemente de su sintomatología, tomamos cultivo de exudado vaginal (CEV) a 103 pacientes; a las otras 97 pacientes lo realizamos si tenían síntomas y/o un pH > 4,5. Solo pusimos tratamiento si eran sintomáticas. Seguimos la evolución de sus embarazos para valorar su patología obstétrica y sus partos. Resultados. La media de pH en la muestra fue 4,41 (4,04–4,78). Encontramos una asociación estadísticamente muy significativa entre la elevación de pH vaginal superior a 4,5 y el aislamiento mediante cultivo de Gardnerella vaginalis (G. vaginalis) (p<0,000), con un OR de 35,15 (4,67–730,39). La mayoría de los cultivos fueron normales, y los gérmenes aislados por orden de frecuencia fueron: Candida albicans (C. albicans) (17,5%), G. vaginalis (7,8%) y Streptococcus agalactiae (7,8%), siendo más del 85% de las gestantes portadoras asintomáticas. La patología obstétrica registrada en nuestra muestra ha sido: amenazas de parto prematuro (2%), roturas prematuras de membranas (2%), partos pretérmino (4,5%) y abortos espontáneos (6%). Conclusiones. El pH vaginal puede ser una buena prueba de cribado para el diagnóstico de vaginosis bacterianas. El germen más prevalente en nuestra muestra ha sido C. albicans siendo la mayoría de gestantes portadoras asintomáticas. No hemos encontrado relación entre la patología obstétrica registrada y los gérmenes aislados en los cultivos de exudado vaginal (AU)

Objective. To assess the usefulness of vaginal pH determination in the first prenatal visit in the diagnosis of vulvovaginitis and its association with obstetric abnormalities. Material and methods. We determined vaginal pH in the first prenatal visit in 200 low-risk pregnancies. In 103 women, vaginal secretions were cultured, independently of their symptoms. In the remaining 97 women, culture was performed only if symptoms were present and/or pH was >4.5. Treatment was prescribed in symptomatic women only. The pregnancies were followed up to evaluate obstetric abnormalities and data on deliveries. Results. The mean pH in the study sample was 4.41 (4.04–4.78). We found a statistically significant association (P<.000) between an increase in vaginal pH over 4.5 and isolation through culture of Gardnerella vaginalis (G. vaginalis), with an OR of 35.15 (4.67–730.39). Most cultures were normal. In order of frequency, the identified microorganisms were Candida albicans (C. albicans) (17.5%), G. vaginalis (7.8%) and Streptococcus agalactiae (7.8%). More than 85% of the carriers were asymptomatic. Obstetric pathology in our sample consisted of preterm labor (2%), premature rupture of membranes (2%), preterm delivery (4.5%) and miscarriage (6%). Conclusions. Vaginal pH determination might be an effective screening test for the diagnosis of bacterial vaginosis. The most prevalent microorganism in our sample was C. albicans and most carriers were asymptomatic. We found no relationship between obstetric pathology and the microorganisms isolated in cultures of vaginal secretions (AU)

Evolution of hepatitis B virus during long-term therapy in patients with chronic hepatitis B.

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.

Construction and phenotypic characterization of HIV type 1 functional envelope clones of subtypes G and F.

Subtype G has been estimated to represent the fourth most prevalent clade in the HIV-1 pandemic and subtype F is widely circulating in parts of South America (frequently within BF recombinant forms) and in Romania. However, functional envelope clones of these subtypes are lacking, which are needed for studies on antibody-mediated neutralization, coreceptor usage, and efficiency of viral entry inhibitor drugs. Here we report the construction, neutralization properties, and coreceptor usage of HIV-1 functional envelope clones of subtypes G (n = 15) and F (n = 7). These clones were obtained through RT-PCR amplification of HIV-1 gp160 from plasma RNA, and were used for pseudovirus production. All 15 subtype G-enveloped pseudoviruses were resistant to neutralization by gp120-targeted broadly neutralizing monoclonal antibodies (MAbs) b12 and 2G12, while a majority were neutralized by gp41-targeted MAbs 2F5 and 4E10. With regard to the subtype F envelopes, all seven pseudoviruses were resistant to 2F5 and b12, six were resistant to G12, and six were neutralized by 4E10. Coreceptor usage testing revealed that 21 of 22 envelopes were CCR5-tropic, including all 15 subtype G envelopes, seven of which were from patients with CD4(+) T cell counts <200/ml. These results confirm the broadly neutralizing activity of 4E10 on envelope clones across all tested group M clades, including subtypes G and F, reveal the resistance of most subtype F-enveloped pseudoviruses to broadly neutralizing MAbs b12, 2G12, and 2F5, and suggest that, similarly to subtype C, CXCR4 tropism is uncommon in subtype G, even at advanced stages of infection.