[Plasmid-mediated AMPc producing Proteus mirabilis in the Health Care Area of Santiago de Compostela: molecular and epidemiological analysis by rep-PCR and MALDI-TOF]. / Proteus mirabilis productor de AmpC plasmídica en el Área Sanitaria de Santiago de Compostela: prevalencia y caracterización molecular por rep-PCR y MALDI-TOF MS.

INTRODUCTION: Proteus mirabilis is an important pathogen isolated from both community-acquired and health-care associated infections. Acquired AmpC-type beta-lactamases represent an important mechanism of resistance to extended-spectrum cephalosporins and are emerging in several European countries. The objective of this work was to know the prevalence of acquired AmpC beta-lactamase producing P. mirabilis over the last three years and eight months and their clonal relationships comparing MALDI-TOF and automated rep-PCR results. METHODS: P. mirabilis isolates (n= 1,396) were obtained from routine cultures at the University Hospital Complex of Santiago de Compostela from January 2006 to August 2009. Identification to the species level and antimicrobial susceptibility testing were achieved with Vitek 2. The isolates showing intermediate or total resistance to amoxicillin-clavulanic and cefoxitin, cefotaxime or ceftazidime were selected for AmpC phenotypic detection by double-disk synergy test, and molecular confirmation by multiplex PCR. Molecular typing of the isolates was performed by automated rep-PCR and MALDI-TOF. RESULTS: For the last three years and eight months, the prevalence of AmpC-producing P. mirabilis increased from 0.17% to 4.5%, mainly associated with urinary tract infection in elderly outpatients. In all cases, plasmidic AmpC belonging to LAT/CMY lineage were detected. A high genetic variability was seen with both, rep-PCR and MALDI-TOF MS. CONCLUSIONS: AmpC-producing P. mirabilis is an emergent pathogen. The high genetic variability detected suggests that the spread of the resistance mechanism is more probable than a clone dispersion. Automated rep-PCR and MALDI-TOF MS show as fast and decisive methods for bacterial strain typing in clinical microbiology laboratories.

Proteus mirabilis productor de AmpC plasmídica en el Área Sanitaria de Santiago de Compostela: prevalencia y caracterización molecular por rep-PCR y MALDI-TOF MS / Plasmid-mediated AMPc producing Proteus mirabilis in the Health Care Area of Santiago de Compostela: molecular and epidemiological analysis by rep-PCR and MALDI-TOF

Introducción: Proteus mirabilis es un patógeno de importancia creciente tanto en las infecciones nosocomiales como en las comunitarias. La producción de AmpC plasmídica es un mecanismo de resistencia frente a cefalosporinas de espectro extendido emergente en esta bacteria por lo que se consideró de gran interés estudiar su prevalencia en nuestro Área Sanitaria así como su variabilidad genética, comparando dos métodos recientemente incorporados al mercado: MALDI-TOF y rep-PCR automatizada. Métodos: Entre enero de 2006 y agosto de 2009 se recuperaron 1.396 aislamientos de P. mirabilis a partir de los cultivos de rutina realizados en Complejo Hospitalario Universitario de Santiago de Compostela. La identificación y el antibiograma se hicieron por Vitek 2. Aquellos aislamientos con sensibilidad reducida a amoxicilina-clavulánico y a cefoxitina, cefotaxima o ceftazidima fueron seleccionados para la detección fenotípica y genotípica de AmpC plasmídica mediante sinergia con doble disco y PCR múltiple, respectivamente. La tipificación molecular se llevó a cabo, comparativamente, mediante rep-PCR automatizada y MALDI-TOF. Resultados: A lo largo de tres años y ocho meses, la prevalencia de P. mirabilis productor de AmpC pasó del 0,17% al 4,5%, mayoritariamente asociado a infección urinaria en pacientes ancianos no hospitalizados. En todos los casos, AmpC plasmídica perteneció a la familia LAT/CMY. Se observó una gran variabilidad genética entre los aislamientos tanto por rep-PCR (DiversiLab) como por MALDI-TOF MS. Conclusión: P. mirabilis productor de AmpC adquirida es un patógeno emergente. La variabilidad genética de las cepas estudiadas apunta a una dispersión de este mecanismo de resistencia más que a una diseminación clonal. Rep-PCR automatizada y MALDI-TOF se muestran como métodos rápidos y resolutivos para la tipificación molecular en los laboratorios de microbiología clínica(AU)

Introduction: Proteus mirabilis is an important pathogen isolated from both community-acquired and health-care associated infections. Acquired AmpC-type beta-lactamases represent an important mechanism of resistance to extended-spectrum cephalosporins and are emerging in several European countries. The objective of this work was to know the prevalence of acquired AmpC beta-lactamase producing P. mirabilis over the last three years and eight months and their clonal relationships comparing MALDI-TOF and automated rep-PCR results. Methods: P. mirabilis isolates (n= 1,396) were obtained from routine cultures at the University Hospital Complex of Santiago de Compostela from January 2006 to August 2009. Identification to the species level and antimicrobial susceptibility testing were achieved with Vitek 2. The isolates showing intermediate or total resistance to amoxicillin-clavulanic and cefoxitin, cefotaxime or ceftazidime were selected for AmpC phenotypic detection by double-disk synergy test, and molecular confirmation by multiplex PCR. Molecular typing of the isolates was performed by automated rep-PCR and MALDI-TOF. Results: For the last three years and eight months, the prevalence of AmpC-producing P. mirabilis increased from 0.17% to 4.5%, mainly associated with urinary tract infection in elderly outpatients. In all cases, plasmidic AmpC belonging to LAT/CMY lineage were detected. A high genetic variability was seen with both, rep-PCR and MALDI-TOF MS. Conclusions: AmpC-producing P. mirabilis is an emergent pathogen. The high genetic variability detected suggests that the spread of the resistance mechanism is more probable than a clone dispersion. Automated rep-PCR and MALDI-TOF MS show as fast and decisive methods for bacterial strain typing in clinical microbiology laboratories(AU)

Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF.

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.

Molecular and epidemiological analysis of nosocomial carbapenem-resistant Klebsiella spp. using repetitive extragenic palindromic-polymerase chain reaction and matrix-assisted laser desorption/ionization-time of flight.

INTRODUCTION: Infections with carbapenem-resistant enterobacteria are an emerging threat. This study reports the microbiologic, clinical, and epidemiologic features and the therapeutic outcomes of the infections caused by carbapenem- and pandrug-resistant Klebsiella emerged in our hospital. Fingerprinting analyses by automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry are also compared. MATERIALS AND METHODS: Carbapenem-resistant Klebsiella spp. affecting 13 patients were investigated using automated rep-PCR (DiversiLab System) and MALDI-TOF. Species identification was performed by Vitek 2 System and MALDI-TOF. Antimicrobial susceptibility testing was made using Vitek 2 System and Etest. Screening for extended spectrum beta-lactamase (ESBL) and carbapenemase production was made by double disk synergy and Hodge tests, respectively. Synergy studies were performed using Etest. DNA array was used for detection of KPC and ESBLs. bla(VIM-1) gene was amplified by PCR and sequencing. Use of carbapenems in the hospital was studied. RESULTS: A total of 13 patients were found to be colonized/infected with carbapenem-resistant Klebsiella. All patients were previously submitted to surgery and/or presented with severe underlying disease. After carbapenem-resistant Klebsiella isolation, the majority of the patients were treated with amikacin plus carbapenem, tigecycline, or fosfomycin. All Klebsiella isolates (n = 14), except two, had the bla(VIM-1) gene and all Klebsiella pneumoniae also had bla(SHV) gene associated with ESBL production. DiversiLab system showed higher discriminatory power than MALDI-TOF for strain typing. CONCLUSIONS: The risk of a rapid dissemination and the persistence of these multidrug-resistant strains through the time determine the need to implement routine procedures for metallo-beta-lactamase detection and measures for prevention of the spread of these microorganisms. The combined use of MALDI-TOF for species identification and DiversiLab System for clonal strain typing may be a useful tool for fast and accurate management of nosocomial outbreaks. The potential clinical utility of fosfomycin in this matter should be considered in future studies.

Automated extraction improves multiplex molecular detection of infection in septic patients.

Sepsis is one of the leading causes of morbidity and mortality in hospitalized patients worldwide. Molecular technologies for rapid detection of microorganisms in patients with sepsis have only recently become available. LightCycler SeptiFast test M(grade) (Roche Diagnostics GmbH) is a multiplex PCR analysis able to detect DNA of the 25 most frequent pathogens in bloodstream infections. The time and labor saved while avoiding excessive laboratory manipulation is the rationale for selecting the automated MagNA Pure compact nucleic acid isolation kit-I (Roche Applied Science, GmbH) as an alternative to conventional SeptiFast extraction. For the purposes of this study, we evaluate extraction in order to demonstrate the feasibility of automation. Finally, a prospective observational study was done using 106 clinical samples obtained from 76 patients in our ICU. Both extraction methods were used in parallel to test the samples. When molecular detection test results using both manual and automated extraction were compared with the data from blood cultures obtained at the same time, the results show that SeptiFast with the alternative MagNA Pure compact extraction not only shortens the complete workflow to 3.57 hrs., but also increases sensitivity of the molecular assay for detecting infection as defined by positive blood culture confirmation.

Comparación entre las pruebas para la detección de betalactamasas de espectro extendido de los sistemas Vitek 2 y Phoenix / Comparative assessment of the Vitek 2 and Phoenix systems for detection of extended-spectrum beta-lactamases

Introducción La detección de la multirresistencia a betalactámicos en Escherichia coli y Klebsiella pneumoniae es una cuestión clínicamente relevante. Por otro lado, es interesante diferenciar entre la producción de betalactamasas de espectro extendido (BLEE) y otros mecanismos de resistencia para evitar el tratamiento inadecuado de infecciones causadas por este tipo de cepas. El objetivo del presente estudio fue comparar la capacidad de las pruebas confirmatorias de los sistemas automatizados Vitek 2 y Phoenix para detectar la producción de BLEE en E. coli y K. pneumoniae. Material y métodos Se ensayaron 193 aislamientos clínicos fenotípicamente productores de BLEE (174 E. coli y 19K. pneumoniae) por Vitek 2 y BD Phoenix System y se utilizaron las tarjetas AST-N058 y los paneles UNMIC/ID-62, respectivamente. Se consideraron métodos fenotípicos de referencia la prueba de sinergia con doble disco y Etest. Como controles positivos y negativos se ensayaron 12 cepas genotípicamente caracterizadas. Resultados En el caso de los aislamientos clínicos, la sensibilidad fue del 99,5% para Vitek 2 y del 95,3% para Phoenix. En las cepas control no hubo diferencias entre ambos sistemas. La ejecución del sistema experto elevó la sensibilidad del Phoenix al 100%. Sin embargo, el sistema experto de Vitek 2 consideró incoherentes los resultados obtenidos en 7 aislamientos con la prueba para BLEE positiva. Conclusión La sensibilidad de la prueba confirmatoria para la producción de BLEE es superior en las tarjetas N-058 de Vitek. No obstante, la actuación de los sistemas expertos sitúa a ambos sistemas a la misma altura en su capacidad de detección de BLEE en E. coli y K. pneumoniae (AU)

Introduction and Purpose Detection of beta-lactam resistance in Escherichia coli and Klebsiella pneumoniae strains is clinically relevant. Moreover, it is important to differentiate between extended-spectrum beta-lactamase (ESBL) production and other mechanisms of resistance to avoid inadequate treatment of infection caused by these strains. The aim of this study was to compare the performance of the Vitek 2 and BD Phoenix automated systems for confirmatory testing of ESBL production. Material and Methods A total of 193 clinical isolates of phenotypically confirmed ESBL producers (174 E. coli and 19 K. pneumoniae) were assayed by the Vitek 2 and BD Phoenix systems using AST-N058 cards and UNMIC/ID-62 panels, respectively. The double-disk synergy test and the Etest were used as phenotype reference methods. Twelve strains characterized by genotyping were used as positive and negative controls. Results In the clinical isolates, the sensitivity of the tests was 99.5% for Vitek and 95.3% for Phoenix. There were no significant differences between the 2 systems in the control strains. Execution of the expert system raised the sensitivity of Phoenix to 100%. However, the Vitek 2 expert system considered the results obtained in 7 strains with ESBL-positive tests to be incoherent. Conclusion Confirmatory testing for ESBL production with the Vitek 2 system (AST-N058 card) showed higher sensitivity than the Phoenix (UNMIC-ID 62 panel) system. Nevertheless, the performance of the expert systems in the 2 automated tests was similar for ESBL detection in E. coli and K. pneumonia (AU)

Potential clinical utility of polymerase chain reaction in microbiological testing for sepsis.

OBJECTIVES: To evaluate the potential improvement of antimicrobial treatment by utilizing a new multiplex polymerase chain reaction (PCR) assay that identifies sepsis-relevant microorganisms in blood. DESIGN: Prospective, observational international multicentered trial. SETTING: University hospitals in Germany (n = 2), Spain (n = 1), and the United States (n = 1), and one Italian tertiary general hospital. PATIENTS: 436 sepsis patients with 467 episodes of antimicrobial treatment. METHODS: Whole blood for PCR and blood culture (BC) analysis was sampled independently for each episode. The potential impact of reporting microorganisms by PCR on adequacy and timeliness of antimicrobial therapy was analyzed. The number of gainable days on early adequate antimicrobial treatment attributable to PCR findings was assessed. MEASUREMENTS AND MAIN RESULTS: Sepsis criteria, days on antimicrobial therapy, antimicrobial substances administered, and microorganisms identified by PCR and BC susceptibility tests. RESULTS: BC diagnosed 117 clinically relevant microorganisms; PCR identified 154. Ninety-nine episodes were BC positive (BC+); 131 episodes were PCR positive (PCR+). Overall, 127.8 days of clinically inadequate empirical antibiotic treatment in the 99 BC+ episodes were observed. Utilization of PCR-aided diagnostics calculates to a potential reduction of 106.5 clinically inadequate treatment days. The ratio of gainable early adequate treatment days to number of PCR tests done is 22.8 days/100 tests overall (confidence interval 15-31) and 36.4 days/100 tests in the intensive care and surgical ward populations (confidence interval 22-51). CONCLUSIONS: Rapid PCR identification of microorganisms may contribute to a reduction of early inadequate antibiotic treatment in sepsis.

[Comparative assessment of the Vitek 2 and Phoenix systems for detection of extended-spectrum beta-lactamases]. / Comparación entre las pruebas para la detección de betalactamasas de espectro extendido de los sistemas Vitek 2 y Phoenix.

INTRODUCTION AND PURPOSE: Detection of beta-lactam resistance in Escherichia coli and Klebsiella pneumoniae strains is clinically relevant. Moreover, it is important to differentiate between extended-spectrum beta-lactamase (ESBL) production and other mechanisms of resistance to avoid inadequate treatment of infection caused by these strains. The aim of this study was to compare the performance of the Vitek 2 and BD Phoenix automated systems for confirmatory testing of ESBL production. MATERIAL AND METHODS: A total of 193 clinical isolates of phenotypically confirmed ESBL producers (174 E. coli and 19 K. pneumoniae) were assayed by the Vitek 2 and BD Phoenix systems using AST-N058 cards and UNMIC/ID-62 panels, respectively. The double-disk synergy test and the Etest were used as phenotype reference methods. Twelve strains characterized by genotyping were used as positive and negative controls. RESULTS: In the clinical isolates, the sensitivity of the tests was 99.5% for Vitek and 95.3% for Phoenix. There were no significant differences between the 2 systems in the control strains. Execution of the expert system raised the sensitivity of Phoenix to 100%. However, the Vitek 2 expert system considered the results obtained in 7 strains with ESBL-positive tests to be incoherent. CONCLUSION: Confirmatory testing for ESBL production with the Vitek 2 system (AST-N058 card) showed higher sensitivity than the Phoenix (UNMIC-ID 62 panel) system. Nevertheless, the performance of the expert systems in the 2 automated tests was similar for ESBL detection in E. coli and K. pneumoniae.

Implication of p38 mitogen-activated protein kinase isoforms (alpha, beta, gamma and delta) in CD4+ T-cell infection with human immunodeficiency virus type I.

The CD4(+) T-cell reduction characteristic of human immunodeficiency virus type 1 (HIV-1) infection is thought to result, in addition to infected T-cell death, mainly from uninfected bystander T-cell apoptosis. Nevertheless, the immunological and virological mechanisms leading to T-cell death during HIV-1 infection are not yet fully understood. In the present study, we analysed the individual implication of the p38 mitogen-activated protein kinase (MAPK) isoforms (p38alpha, p38beta, p38gamma and p38delta) during apoptosis induced by HIV-1, taking into account that HIV-1 replication is known to be blocked by p38 inhibitors. For this purpose, we used the SupT1 cell line, where death induced by HIV-1 mainly occurs by uninfected bystander cell apoptosis. A variety of SupT1-based cell lines were constructed constitutively expressing, under the control of cytomegalovirus promoter (PCMV), each dominant-negative (dn) p38 isoform and each wild-type p38 isoform as a control. An enhanced green fluorescent protein marker gene, under the control of the HIV-1 promoter, was inserted in all of them. These cell lines were infected with HIV-1 and analysed by flow cytometry. We found that survival in SupT1-based cell lines infected by HIV-1 was increased by the p38alphadn, p38gammadn and p38deltadn isoforms, but not by the p38betadn isoform. HIV-1 replication was delayed most by p38deltadn and to a lesser extent by p38alphadn and p38gammadn. Moreover, these three isoforms, p38alphadn, p38gammadn and p38deltadn, reduced apoptosis induced by HIV-1. These results suggest that, in SupT1-based cell lines, p38alpha, p38gamma and p38delta, but not p38beta, are implicated in both HIV-1 induced replication and apoptosis in infected and uninfected bystander cells.

[Epidemiology and clinical manifestations of viral hepatitis]. / Epidemiología y manifestaciones clínicas de las hepatitis virales.

Viral hepatitis is an infectious disease affecting the liver. At this time, five different human hepatitis viruses are recognized and characterized in detail: Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). The hepatitis viruses differ widely in their modes of transmission and clinical features. Whereas all of them can cause acute hepatitis, only HBV, HDV, and HCV cause chronic hepatitis. This article reviews the epidemiology and clinical manifestations of the different types of viral hepatitis.

Epidemiología y manifestaciones clínicas de las hepatitis virales / Epidemiology and clinical manifestations of viral hepatitis

La hepatitis viral es una enfermedad infecciosa que afecta al hígado. Hasta el momento, han sido reconocidos y caracterizados minuciosamente en humanos cinco diferentes virus de la hepatitis: el virus de la hepatitis A (VHA), el virus de la hepatitis B (VHB), el virus de la hepatitis C (VHC), el virus de la hepatitis D (VHD) y el virus de la hepatitis E (VHE). Los virus de la hepatitis difieren ampliamente en sus formas de transmisión y características clínicas; ya que mientras todos pueden causar hepatitis aguda, sólo el VHB, el VHD y el VHC ocasionan hepatitis crónica. Este artículo presenta una revisión sobre la epidemiología y manifestaciones clínicas de las diferentes hepatitis virales (AU)

Viral hepatitis is an infectious disease affecting the liver. At this time, five different human hepatitis viruses are recognized and characterized in detail: Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). The hepatitis viruses differ widely in their modes of transmission and clinical features. Whereas all of them can cause acute hepatitis, only HBV, HDV, and HCV cause chronic hepatitis. This article reviews the epidemiology and clinical manifestations of the different types of viral hepatitis (AU)

The future of HIV infection: gene therapy and RNA interference.

The description of the mechanism of RNA interference (RNAi) has generated enormous interest in the biomedical field. A previously unrecognized pathway in which small interfering, 21 to 23 mer, double-stranded RNA (siRNA) mediates sequence-specific degradation of mRNA is becoming one the most useful techniques in cell biology and genetics research. Based on the potency, specificity and physiology of RNAi to silence gene expression, much is expected from its use as a therapeutic tool. The first evidence of RNAi as a suppressor of HIV replication has already been reported, thus providing a new impetus to the development of molecular or gene therapy approaches to HIV infection.

El futuro en la infección por VIH: terapia génica y ARN de interferencia / The Future in HIV infection: Gene Therapy and RNA interference

La descripción del mecanismo de interferencia mediada por ARN (ARNi) ha despertado un enorme interés en todos los campos de la biomedicina. Una vía previamente desconocida en la que fragmentos de doble hebra de ARN de 21 a 23 residuos (ARNpi) median la degradación específica de secuencias de ARNm se está convirtiendo en una de las herramientas más poderosas en la investigación en biología celular y genética. Existen grandes expectativas en el uso terapéutico silenciamiento genético por medio de ARNi que se basan en su potencia, especificidad y fisiología. La comunicación de las primeras evidencias sobre la efectividad del ARNi como supresor de la replicación de VIH ha estimulado de nuevo el desarrollo de estrategias de terapia génica o molecular para el tratamiento de la infección por VIH

The description of the mechanism of RNA interference (RNAi) has generated enormous interest in the biomedical field. A previously unrecognized pathway in which small interfering, 21 to 23 mer, double-stranded RNA (siRNA) mediates sequence-specific degradation of mRNA is becoming one the most useful techniques in cell biology and genetics research. Based on the potency, specificity and physiology of RNAi to silence gene expression, much is expected from its use as a therapeutic tool. The first evidence of RNAi as a suppressor of HIV replication has already been reported, thus providing a new impetus to the development of molecular or gene therapy approaches to HIV infection

Construction and characterization of a minimized version of the HIV-1 pNL4-3 plasmid and its application for pseudotyping HIV-1 vectors.

The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions. The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL4-3 digestion by using restriction sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence, (3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps 2 and 3, (5) ligation of the fragment containing ColE1+Amp(R) with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally used cotransfection method.

Hepatitis B virus genotyping based on cluster analysis of the region involved in lamivudine resistance.

Hepatitis B virus (HBV) genotyping and treatment resistances analysis provide key information for the study of infected patients. Among the existing HBV genotyping methods, restriction fragment length polymorphism (RFLP) based methods are used widely, but HBV genetic variability may lead to wrong results. A simple method for HBV genotyping is described. This single assay provides information related to both genotyping and lamivudine resistance. measuring genotyping reliability. A short region including the YMDD motif of the polymerase gene was analyzed using cluster analysis of 55 isolates. To discriminate the seven HBV genotypes, a selected fragment was used as representative of each genotype (consensus sequences). Comparison between complete genomes from GenBank and from the YMDD analysis using PHILIP package was used for method testing. Sequencing of 102 different serum samples was carried out, and the results were compared with representative sequences. Consensus sequences were chosen corresponding to the different HBV genotypes. Statistical comparison between our method and others gives more than 90% of coincidences with a critical level of 0.056. Comparison between RFLP and the method described gives 3% of discordance results and 3% of untypables samples by RFLP. All the discordant results observed had a change in the pre-S sequence which introduced a new restriction site.