Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens.

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.

Molecular diagnosis of bloodstream infections in onco-haematology patients with PCR/ESI-MS technology.

OBJECTIVES: Onco-haematological patients are prone to develop infections, and antibiotic prophylaxis may lead to negative blood cultures. Thus, the microbiological diagnosis and subsequent administration of a targeted antimicrobial therapy is often difficult. The goal of this study was to evaluate the usefulness of IRIDICA (PCR/ESI-MS technology) for the molecular diagnosis of bloodstream infections in this patient group. METHODS: A total of 463 whole blood specimens from different sepsis episodes in 429 patients were analysed using the PCR/ESI-MS platform, comparing the results with those of blood culture and other clinically relevant information. RESULTS: The sensitivity of PCR/ESI-MS by specimen (excluding polymicrobial infections, n = 25) in comparison with blood culture was 64.3% overall, 69.0% in oncological patients, and 59.3% in haematological patients. When comparing with a clinical infection criterion, overall sensitivity rose to 74.7%, being higher in oncological patients (80.0%) than in haematological patients (67.7%). Thirty-one microorganisms isolated by culture were not detected by IRIDICA, whereas 42 clinically relevant pathogens not isolated by culture were detected moleculary. CONCLUSIONS: PCR/ESI-MS offers a reliable identification of pathogens directly from whole blood. While additional studies are needed to confirm our findings, the system showed a lower sensitivity in onco-haematological patients in comparison with previously reported results in patients from the Intensive Care Unit.

Usefulness of a novel multiplex real-time PCR assay for the diagnosis of sexually-transmitted infections / Utilidad de un nuevo ensayo de PCR multiplex para el diagnóstico de las infecciones de transmisión sexual

INTRODUCTION: Sexually transmitted infections (STI) are currently on the increase worldwide. New molecular tools have been developed in the past few years in order to improve their diagnosis. An evaluation was carried out using a new commercially available real-time PCR assay, Anyplex™ II STI-7 (Seegene, Seoul, Korea), which detects seven major pathogens in a single reaction - Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Ureaplasma parvum - and compared with conventional methods performed in our laboratory. MATERIALS AND METHODS: Two different populations were included, and 267 specimens from different sites of infection (urines, endocervical swabs, rectal swabs, vaginal swabs, urethral swabs and one inguinal adenopathy) were processed for both methods. RESULTS: The parameters of clinical performance were calculated for C. trachomatis, N. gonorrhoeae, and T. vaginalis, and the assay achieved sensitivities (SE) from 93.94% to 100%, and specificities (SP) from 96.55% to 100%, with negative predictive values (NPV) from 93.33% to 98.85%, and positive predictive values (PPV) from 96.88% to 100%, with a very good agreement (kappa index from 0.88 to 1). CONCLUSIONS: Anyplex™ II STI-7 is a good tool for the reliable diagnosis of STI. Its ease of use and processing allows it to be incorporated into the day to day laboratory work

INTRODUCCIÓN: Las infecciones de transmisión sexual (ITS) son actualmente un problema de salud pública en todo el mundo debido al aumento que han experimentado en los últimos años que implica el desarrollo de nuevas herramientas moleculares para mejorar su diagnóstico. Se ha comparado el nuevo ensayo de PCR en tiempo real, Anyplex™ II STI-7 (Seegene, Seúl, Corea) que detecta los siete microorganismos implicados en las ITS - Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Ureaplasma parvum - en una sola reacción, con los métodos convencionales utilizados en nuestro laboratorio. MÉTODOS: Se incluyeron dos tipos de poblaciones, obteniéndose 267 muestras de diferentes lugares de infección (orines, exudados endocervicales, frotis rectales, frotis vaginales, exudados uretrales y una adenopatía inguinal) que fueron procesadas por ambas metodologías. RESULTADOS: Las sensibilidades, especificidades y valores predictivos fueron analizados para C. trachomatis, N. gonorrhoeae y T. vaginalis, alcanzando sensibilidades (SE) de 93,94% a 100%, especificidades (SP) de 96,55% a 100%, valor predictivo negativo (NPV) entre 93,33% y el 98,85% y valores predictivos positivos (PPV) de 96.88% a 100% con muy buena correlación (índice kappa de 0.88 a 1). CONCLUSIONES: AnyplexTM II STI-7 es una buena herramienta para el diagnóstico seguro de las ITS. La facilidad de uso y procesamiento permite su incorporación en el trabajo del día a día del laboratorio

Identification of hepatitis C virus genotype 3 by a commercial assay challenged by natural polymorphisms detected in Spain from patients with diverse origins.

BACKGROUND: Hepatitis C virus (HCV) genotyping is crucial in clinical practise for determining the type and duration of antiviral therapy. Between 2009 and 2014, 24 (7.95%) of all HCV genotype 3 (HCV-3) cases obtained indeterminate results via the RealTime HCV Genotype II assay (Abbott) at a tertiary care center in Spain. HCV-3 is the second most common genotype worldwide. Moreover, it has been associated with a higher risk of liver disease progression and a lower response to the latest antivirals. OBJECTIVE: Given the clinical significance of accurately identifying HCV-3, we aimed to characterize the genetic diversity of the HCV 5' untranslated region (5' UTR), the target of genotyping assays, by ultradeep pyrosequencing (UDPS). STUDY DESIGN: For the 24 indeterminate samples, the 5' UTR-core was amplified and subjected to UDPS with the 454/GS-Junior platform (Roche). The genotype/subtype of each identified haplotype was assigned by phylogenetic analysis. For comparison, three additional samples correctly identified as HCV-3 by the real-time assay were also analyzed. RESULTS: HCV genotyping based on 5' UTR-core UDPS was in agreement with NS5B Sanger sequencing in all cases, confirming the absence of mixed infections and recombination events. The generated 5' UTR sequences proved the presence of one to three polymorphisms at the probe-binding site of the Abbott assay, thereby differentiating indeterminate from correctly genotyped HCV-3 samples. CONCLUSIONS: The observed naturally occurring polymorphisms provide insight into regional differences observed with genotype 3, their impact on genotyping assay performance, and potential improvement and designing options.

Evaluation of the SediMax automated microscopy sediment analyzer and the Sysmex UF-1000i flow cytometer as screening tools to rule out negative urinary tract infections.

Urinary tract infections (UTI) are highly prevalent in nosocomial and community settings, and their diagnosis is costly and time-consuming. Screening methods represent an important advance towards the final UTI diagnosis, diminishing inappropriate treatment or clinical complications. Automated analyzers have been developed and commercialized to screen and rule out negative urine samples. The aim of this study was to evaluate two of these automated analyzers (SediMax, an automatic sediment analyzer and UF-1000i a flow cytometer) to predict negative urine cultures. A total of 1934 urine samples were analyzed. A very strong correlation for white blood cells (WBC) (rs: 0.928) and a strong correlation for bacteria (BAC) (rs: 0.693) were obtained. We also calculated optimal cut-off points for both autoanalyzers: 18 WBC/µL and 97 BAC/µL for SediMax (sensitivity=96.25%, specificity=63.04%, negative predictive value=97.97%), and 40 WBC/µL and 460 BAC/µL for UF-1000i (sensitivity=98.13%, specificity=79.16%, negative predictive value=99.18%). The use of SediMax and UF-1000i resulted in a 46.33% and 57.19% reduction of all samples cultured, respectively. In conclusion, both analyzers are good UTI screening tools in our setting.

Direct Identification of Urinary Tract Pathogens from Urine Samples, Combining Urine Screening Methods and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Early diagnosis of urinary tract infections (UTIs) is essential to avoid inadequate or unnecessary empirical antibiotic therapy. Microbiological confirmation takes 24 to 48 h. The use of screening methods, such as cytometry and automated microscopic analysis of urine sediment, allows the rapid prediction of negative samples. In addition, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a widely established technique in clinical microbiology laboratories used to identify microorganisms. We evaluated the ability of MALDI-TOF MS to identify microorganisms from direct urine samples and the predictive value of automated analyzers for the identification of microorganisms in urine by MALDI-TOF MS. A total of 451 urine samples from patients with suspected UTIs were first analyzed using the Sysmex UF-1000iflow cytometer, an automatic sediment analyzer with microscopy (SediMax), culture, and then processed by MALDI-TOF MS with a simple triple-centrifuged procedure to obtain a pellet that was washed and centrifuged and finally applied directly to the MALDI-TOF MS plate. The organisms in 336 samples were correctly identified, mainly those with Gram-negative bacteria (86.10%). No microorganisms were misidentified, and noCandidaspp. were correctly identified. Regarding the data from autoanalyzers, the best bacteriuria cutoffs were 1,000 and 200 U/µl for UF-1000iand SediMax, respectively. It was concluded that the combination of a urine screening method and MALDI-TOF MS provided a reliable identification from urine samples, especially in those containing Gram-negative bacteria.

Usefulness of a novel multiplex real-time PCR assay for the diagnosis of sexually-transmitted infections.

INTRODUCTION: Sexually transmitted infections (STI) are currently on the increase worldwide. New molecular tools have been developed in the past few years in order to improve their diagnosis. An evaluation was carried out using a new commercially available real-time PCR assay, Anyplex™ II STI-7 (Seegene, Seoul, Korea), which detects seven major pathogens in a single reaction - Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Ureaplasma parvum - and compared with conventional methods performed in our laboratory. MATERIALS AND METHODS: Two different populations were included, and 267 specimens from different sites of infection (urines, endocervical swabs, rectal swabs, vaginal swabs, urethral swabs and one inguinal adenopathy) were processed for both methods. RESULTS: The parameters of clinical performance were calculated for C. trachomatis, N. gonorrhoeae, and T. vaginalis, and the assay achieved sensitivities (SE) from 93.94% to 100%, and specificities (SP) from 96.55% to 100%, with negative predictive values (NPV) from 93.33% to 98.85%, and positive predictive values (PPV) from 96.88% to 100%, with a very good agreement (kappa index from 0.88 to 1). CONCLUSIONS: Anyplex™ II STI-7 is a good tool for the reliable diagnosis of STI. Its ease of use and processing allows it to be incorporated into the day to day laboratory work.

Evaluation of the Broad-Range PCR/ESI-MS Technology in Blood Specimens for the Molecular Diagnosis of Bloodstream Infections.

BACKGROUND: Rapid identification of the etiological agent in bloodstream infections is of vital importance for the early administration of the most appropriate antibiotic therapy. Molecular methods may offer an advantage to current culture-based microbiological diagnosis. The goal of this study was to evaluate the performance of IRIDICA, a platform based on universal genetic amplification followed by mass spectrometry (PCR/ESI-MS) for the molecular diagnosis of sepsis-related pathogens directly from the patient's blood. METHODS: A total of 410 whole blood specimens from patients admitted to Emergency Room (ER) and Intensive Care Unit (ICU) with clinical suspicion of sepsis were tested with the IRIDICA BAC BSI Assay (broad identification of bacteria and Candida spp.). Microorganisms grown in culture and detected by IRIDICA were compared considering blood culture as gold standard. When discrepancies were found, clinical records and results from other cultures were taken into consideration (clinical infection criterion). RESULTS: The overall positive and negative agreement of IRIDICA with blood culture in the analysis by specimen was 74.8% and 78.6%, respectively, rising to 76.9% and 87.2% respectively, when compared with the clinical infection criterion. Interestingly, IRIDICA detected 41 clinically significant microorganisms missed by culture, most of them from patients under antimicrobial treatment. Of special interest were the detections of one Mycoplasma hominis and two Mycobacterium simiae in immunocompromised patients. When ICU patients were analyzed separately, sensitivity, specificity, positive and negative predictive values compared with blood culture were 83.3%, 78.6%, 33.9% and 97.3% respectively, and 90.5%, 87.2%, 64.4% and 97.3% respectively, in comparison with the clinical infection criterion. CONCLUSIONS: IRIDICA is a promising technology that offers an early and reliable identification of a wide variety of pathogens directly from the patient's blood within 6h, which brings the opportunity to improve management of septic patients, especially for those critically ill admitted to the ICU.

Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance.

The aim of this work was to study the diagnostic accuracy of pyrosequencing to detect resistance to fluoroquinolones, kanamycin, amikacin, capreomycin, and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains. One hundred four clinical isolates previously characterized by BACTEC 460TB/MGIT 960 were included. Specific mutations were targeted in gyrA, rrs, eis promoter, and embB. When there was a discordant result between BACTEC and pyrosequencing, Genotype MTBDRsl (Hain Lifescience, Nehren, Germany) was performed. Sensitivity and specificity of pyrosequencing were 70.6% and 100%, respectively, for fluoroquinolones; 93.3% and 81.7%, respectively, for kanamycin; 94.1% and 95.9%, respectively, for amikacin; 90.0% and 100%, respectively, for capreomycin; and 64.8% and 87.8%, respectively, for EMB. This study shows that pyrosequencing may be a useful tool for making early decisions regarding second-line drugs and EMB resistance. However, for a correct management of patients with suspected extensively drug-resistant tuberculosis, susceptibility results obtained by molecular methods should be confirmed by a phenotypic method.

Immunogenicity of 60 novel latency-related antigens of Mycobacterium tuberculosis.

The aim of our work here was to evaluate the immunogenicity of 60 mycobacterial antigens, some of which have not been previously assessed, notably a novel series of in vivo-expressed Mycobacterium tuberculosis (IVE-TB) antigens. We enrolled 505 subjects and separated them in individuals with and without latent tuberculosis infection (LTBI) vs. patients with active tuberculosis (TB). Following an overnight and 7 days stimulation of whole blood with purified recombinant M. tuberculosis antigens, interferon-γ (IFN-γ) levels were determined by ELISA. Several antigens could statistically significantly differentiate the groups of individuals. We obtained promising antigens from all studied antigen groups [dormancy survival regulon (DosR regulon) encoded antigens; resuscitation-promoting factors (Rpf) antigens; IVE-TB antigens; reactivation associated antigens]. Rv1733, which is a probable conserved transmembrane protein encoded in DosR regulon, turned out to be very immunogenic and able to discriminate between the three defined TB status, thus considered a candidate biomarker. Rv2389 and Rv2435n, belonging to Rpf family and IVE-TB group of antigens, respectively, also stood out as LTBI biomarkers. Although more studies are needed to support our findings, the combined use of these antigens would be an interesting approach to TB immunodiagnosis candidates.

Improving the diagnosis of bloodstream infections: PCR coupled with mass spectrometry.

The reference method for the diagnosis of bloodstream infections is blood culture followed by biochemical identification and antibiotic susceptibility testing of the isolated pathogen. This process requires 48 to 72 hours. The rapid administration of the most appropriate antimicrobial treatment is crucial for the survival of septic patients; therefore, a rapid method that enables diagnosis directly from analysis of a blood sample without culture is needed. A recently developed platform that couples broad-range PCR amplification of pathogen DNA with electrospray ionization mass spectrometry (PCR/ESI-MS) has the ability to identify virtually any microorganism from direct clinical specimens. To date, two clinical evaluations of the PCR/ESI-MS technology for the diagnosis of bloodstream infections from whole blood have been published. Here we discuss them and describe recent improvements that result in an enhanced sensitivity. Other commercially available assays for the molecular diagnosis of bloodstream infections from whole blood are also reviewed. The use of highly sensitive molecular diagnostic methods in combination with conventional procedures could substantially improve the management of septic patients.

Tools for the diagnosis of hepatitis C virus infection and hepatic fibrosis staging.

Hepatitis C virus (HCV) infection represents a major public health issue. Hepatitis C can be cured by therapy, but many infected individuals are unaware of their status. Effective HCV screening, fast diagnosis and characterization, and hepatic fibrosis staging are highly relevant for controlling transmission, treating infected patients and, consequently, avoiding end-stage liver disease. Exposure to HCV can be determined with high sensitivity and specificity with currently available third generation serology assays. Additionally, the use of point-of-care tests can increase HCV screening opportunities. However, active HCV infection must be confirmed by direct diagnosis methods. Additionally, HCV genotyping is required prior to starting any treatment. Increasingly, high-volume clinical laboratories use different types of automated platforms, which have simplified sample processing, reduced hands-on-time, minimized contamination risks and human error and ensured full traceability of results. Significant advances have also been made in the field of fibrosis stage assessment with the development of non-invasive methods, such as imaging techniques and serum-based tests. However, no single test is currently available that is able to completely replace liver biopsy. This review focuses on approved commercial tools used to diagnose HCV infection and the recommended hepatic fibrosis staging tests.

Correlation of inflammatory and cardiovascular biomarkers with pneumonia severity scores

PURPOSE: To assess the correlation of procalcitonin (PCT), C-reactive protein (CRP), neopterin, mid-regional pro-atrial natriuretic peptide (MR-proANP), and mid-regional pro-adrenomedullin (MR-proADM) with severity risk scores: severe CAP (SCAP) and SMART-COP in patients with community-acquired pneumonia (CAP), as well as short term prognosis and to determine the correlation with mortality risk scores. METHODS: Eighty-five patients with a final diagnosis of pneumonia were consecutively included during a two month period. Epidemiological, clinical, microbiological, and radiological data were recorded. Patients were stratified according to the PSI, CURB-65, SCAP and SMART-COP. Complications were defined as respiratory failure/shock, need of ICU, and death. Plasma samples were collected at admission. RESULTS: MR-proANP and MR-proADM showed significantly higher levels in high risk SCAP group in comparison to low risk. When considering SMART-COP none of the biomarkers showed statistical differences. MR-proADM levels were high in patients with high risk of needing intensive respiratory or vasopressor support according to SMRT-CO. Neopterin and MR-proADM were significantly higher in patients that developed complications. PCT and MR-proADM showed significantly higher levels in cases of a definite bacterial diagnosis in comparison to probable bacterial, and unknown origin. MR-proANP and MR-proADM levels increased statistically according to PSI and CURB-65. CONCLUSIONS: Biomarker levels are higher in pneumonia patients with a poorer prognosis according to SCAP and SMART-COP indexes, and to the development of complications

OBJETIVO: Establecer la correlación entre los niveles de procalcitonina (PCT), proteína C reactiva, neopterina, pro-péptido natriurético auricular (MR-proANP) y pro-adrenomedulina (MR-proADM) y los índices de severidad: severe CAP (SCAP) y SMART-COP en pacientes con neumonía adquirida en la comunidad (NAC), así como el pronóstico a corto plazo, y confirmar su correlación con los índices de severidad PSI y CURB-65. MÉTODOS: Ochenta y cinco pacientes con diagnóstico final de NAC fueron incluidos de forma consecutiva durante 2 meses. Se recogieron los datos epidemiológicos, clínicos, microbiológicos y radiológicos. Los pacientes se clasificaron en función del PSI, CURB-65, SCAP y SMART-COP. Las complicaciones se definieron como insuficiencia respiratoria/shock, ingreso en la UCI o muerte. Las muestras de plasma se recogieron en el momento del ingreso hospitalario. RESULTADOS: Los niveles de MR-proANP y MR-proADM fueron significativamente superiores en aquellos pacientes clasificados como alto riesgo según SCAP en comparación con los de bajo riesgo. Al considerar SMART-COP ninguno de los biomarcadores mostró significación estadística. Los niveles de MR-proADM fueron superiores en los pacientes con alto riesgo de necesitar soporte intensivo/vasopresor según SMRT-CO. Los valores de neopterina y MR-proADM fueron significativamente superiores en pacientes que desarrollaron alguna complicación. En los casos con diagnóstico bacteriano de seguridad, se observaron niveles significativamente más elevados de PCT y MR-proADM, respecto de los casos de probable origen bacteriano o origen desconocido. Los niveles de MR-proANP y MR-proADM se incrementaron en función del PSI y de CURB-65. CONCLUSIONES: Los niveles de biomarcadores son superiores en pacientes con peor pronóstico, según los índices de severidad evaluados, así como con el desarrollo de complicaciones

Correlation of inflammatory and cardiovascular biomarkers with pneumonia severity scores.

PURPOSE: To assess the correlation of procalcitonin (PCT), C-reactive protein (CRP), neopterin, mid-regional pro-atrial natriuretic peptide (MR-proANP), and mid-regional pro-adrenomedullin (MR-proADM) with severity risk scores: severe CAP (SCAP) and SMART-COP in patients with community-acquired pneumonia (CAP), as well as short term prognosis and to determine the correlation with mortality risk scores. METHODS: Eighty-five patients with a final diagnosis of pneumonia were consecutively included during a two month period. Epidemiological, clinical, microbiological, and radiological data were recorded. Patients were stratified according to the PSI, CURB-65, SCAP and SMART-COP. Complications were defined as respiratory failure/shock, need of ICU, and death. Plasma samples were collected at admission. RESULTS: MR-proANP and MR-proADM showed significantly higher levels in high risk SCAP group in comparison to low risk. When considering SMART-COP none of the biomarkers showed statistical differences. MR-proADM levels were high in patients with high risk of needing intensive respiratory or vasopressor support according to SMRT-CO. Neopterin and MR-proADM were significantly higher in patients that developed complications. PCT and MR-proADM showed significantly higher levels in cases of a definite bacterial diagnosis in comparison to probable bacterial, and unknown origin. MR-proANP and MR-proADM levels increased statistically according to PSI and CURB-65. CONCLUSIONS: Biomarker levels are higher in pneumonia patients with a poorer prognosis according to SCAP and SMART-COP indexes, and to the development of complications.

Relevance of baseline viral genetic heterogeneity and host factors for treatment outcome prediction in hepatitis C virus 1b-infected patients.

BACKGROUND: Only about 50% of patients chronically infected with HCV genotype 1 (HCV-1) respond to treatment with pegylated interferon-alfa and ribavirin (dual therapy), and protease inhibitors have to be administered together with these drugs increasing costs and side-effects. We aimed to develop a predictive model of treatment response based on a combination of baseline clinical and viral parameters. METHODOLOGY: Seventy-four patients chronically infected with HCV-1b and treated with dual therapy were studied (53 retrospectively -training group-, and 21 prospectively -validation group-). Host and viral-related factors (viral load, and genetic variability in the E1-E2, core and Interferon Sensitivity Determining Region) were assessed. Multivariate discriminant analysis and decision tree analysis were used to develop predictive models on the training group, which were then validated in the validation group. PRINCIPAL FINDINGS: A multivariate discriminant predictive model was generated including the following variables in decreasing order of significance: the number of viral variants in the E1-E2 region, an amino acid substitution pattern in the viral core region, the IL28B polymorphism, serum GGT and ALT levels, and viral load. Using this model treatment outcome was accurately predicted in the training group (AUROC = 0.9444; 96.3% specificity, 94.7% PPV, 75% sensitivity, 81% NPV), and the accuracy remained high in the validation group (AUROC = 0.8148, 88.9% specificity, 90.0% PPV, 75.0% sensitivity, 72.7% NPV). A second model was obtained by a decision tree analysis and showed a similarly high accuracy in the training group but a worse reproducibility in the validation group (AUROC = 0.9072 vs. 0.7361, respectively). CONCLUSIONS AND SIGNIFICANCE: The baseline predictive models obtained including both host and viral variables had a high positive predictive value in our population of Spanish HCV-1b treatment naïve patients. Accurately identifying those patients that would respond to the dual therapy could help reducing implementation costs and additional side effects of new treatment regimens.

Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials.

The development of a new tuberculosis vaccine is an urgent need due to the failure of the current vaccine, BCG, to protect against the respiratory form of the disease. MTBVAC is an attenuated Mycobacterium tuberculosis vaccine candidate genetically engineered to fulfil the Geneva consensus requirements to enter human clinical trials. We selected a M. tuberculosis clinical isolate to generate two independent deletions without antibiotic-resistance markers in the genes phoP, coding for a transcription factor key for the regulation of M. tuberculosis virulence, and fadD26, essential for the synthesis of the complex lipids phthiocerol dimycocerosates (DIM), one of the major mycobacterial virulence factors. The resultant strain MTBVAC exhibits safety and biodistribution profiles similar to BCG and confers superior protection in preclinical studies. These features have enabled MTBVAC to be the first live attenuated M. tuberculosis vaccine to enter clinical evaluation.

Rapid diagnosis of bloodstream infections with PCR followed by mass spectrometry.

Achieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood. A total of 247 whole blood samples and paired blood cultures were prospectively obtained from 175 patients with a suspicion of sepsis. Both sample types were analyzed using the PCR/ESI-MS technology, and the results were compared with those obtained by conventional identification methods. The overall agreement between conventional methods and PCR/ESI-MS performed in blood culture aliquots was 94.2% with 96.8% sensitivity and 98.5% specificity for the molecular method. When comparing conventional methods with PCR/ESI-MS performed in whole blood specimens, the overall agreement was 77.1% with 50% sensitivity and 93.8% specificity for the molecular method. Interestingly, the PCR/ESI-MS technology led to the additional identification of 13 pathogens that were not found by conventional methods. Using the PCR/ESI-MS technology the microbiological diagnosis of bloodstream infections could be anticipated in about half of the patients in our setting, including a small but significant proportion of patients newly diagnosed. Thus, this promising technology could be very useful for the rapid diagnosis of sepsis in combination with traditional methods.

Hepatitis C virus transmission during colonoscopy evidenced by phylogenetic analysis.

BACKGROUND: Nosocomial transmission events still play an important role in hepatitis C virus (HCV) spreading. Among most reported medical procedures involved in nosocomial transmission, endoscopy procedures remain controversial and might be underestimated. OBJECTIVE: The aim of the study was to investigate a case of nosocomial person-to-person transmission of HCV in an endoscopy unit. STUDY DESIGN: An acute HCV infection was detected in a person that had undergone a colonoscopy after an HCV-infected patient. Serum samples from both persons were subjected to a molecular epidemiology study. The HCV NS5B genetic region was amplified and directly sequenced and the E1-E2 region was amplified, cloned and sequenced (20 clones per specimen). All sequences were subjected to phylogenetic analyses. A conventional epidemiological investigation was performed to determine the most likely cause of HCV transmission. RESULTS: NS5B sequence analysis revealed that both persons were infected with closely related HCV-1b strains. Furthermore, phylogenetic analysis of E1-E2 sequences evidenced a direct transmission between patients. The epidemiological investigation pointed out to anesthetic procedures as the most likely source of HCV transmission. The index case, not having spontaneously cleared the infection 10 months after infection, required antiviral treatment, which resulted in a sustained virological response. CONCLUSIONS: The molecular epidemiology study performed provided evidence of a person-to-person transmission of HCV during a colonoscopy procedure, and the anesthetic procedure was the most likely source of HCV transmission. This study highlights the importance of strictly following standard precautions by healthcare workers in order to prevent nosocomial HCV transmission.

Evolutionary dynamics of the E1-E2 viral populations during combination therapy in non-responder patients chronically infected with hepatitis C virus subtype 1b.

Half of the patients chronically infected with hepatitis C virus (HCV) genotype 1 fail to respond to pegylated interferon alpha (PEG-IFN) and ribavirin (RBV) therapy. This study assesses the effects of treatment on the evolution of the E1-E2 viral region in non-responder patients infected with HCV-1b. Twenty-three HCV-1b chronically infected patients were studied retrospectively, including 19 non-responders to PEG-IFN/RBV therapy (11 null-responders and 8 relapsers) in the study group, and 4 untreated patients in the control group. Genetic and phylogenetic analyses of the E1-E2 viral populations were performed at baseline and at the time of treatment failure to assess changes in genetic variability and evolutionary dynamics during treatment. Baseline virological characteristics were similar in null-responders, relapsers and controls. E1-E2 genetic variability decreased during treatment in non-responders, with a more pronounced decline in relapsers than in null-responders. A specific evolutionary pattern was not observed in null-responders, while a complete substitution of viral variants found at baseline characterised relapser patients. No specific E1-E2 amino acid substitution involved in treatment failure could be identified. In conclusion, although diverse evolutionary patterns with no apparent common adaptive changes were observed during therapy, treatment failure was characterised by a decline in genetic diversity.

Comparison between two human papillomavirus genotyping assays targeting the L1 or E6/E7 region in cervical cancer biopsies

Introduction: Human papillomavirus (HPV) testing is increasingly used in cervical cancer prevention strategies, and a variety of HPV genotyping assays have been developed. We aimed to compare the performance of two HPV genotyping techniques in formalin-fixed paraffin-embedded (FFPE) tissue specimens from a series of invasive squamous cell carcinoma (SCC) cases. Methods Archival FFPE tissue blocks from 78 SCC cases were initially considered. DNA was extracted from dewaxed tissue sections and tested with the INNO-LiPA HPV Genotyping Extra assay (Innogenetics), and the F-HPV typing kit (Genomed) targeting the L1 and E6/E7 regions, respectively. Results The INNO-LiPA assay showed a higher sensitivity (98.6%) than the F-HPV assay (78.6%). A total of 12 (17.1%) biopsies showed multiple-type infections evidenced by at least one assay. Among the SCC cases tested, HPV16 and/or 18 were detected in 70% of the cases, and 18.4% of them had multiple infections with other high-risk types. Conclusions Our results suggest that the INNO-LiPA assay has a better performance than the F-HPV in FFPE specimens, probably due to its smaller amplicon size and the wider range of detectable HPV types. The prevalence of multiple infections could be higher than previously reported, as evidenced by the combination of the two assays (AU)

Introducción: La detección del virus del papiloma humano (VPH) es cada vez más utilizada en los algoritmos de prevención del cáncer cervical, y se ha desarrollado una gran variedad de ensayos para su detección y genotipado. Nuestro objetivo fue comparar dos técnicas de genotipado del VPH en muestras de tejido (..) (AU)