Usefulness of matrix-assisted laser desorption ionization/time of flight mass spectrometry for the identification of Streptococcus mutans.

This study evaluated the reliability of MALDI-TOF MS coupled with statistical tools for the identification of Streptococcus mutans in comparison with PCR-based techniques. Bacterial isolates were identified and serotyped by conventional PCR, using S. mutans species and serotype-specific primers. For bacterial identification, mass spectra data from S. mutans and other streptococci were compared with Biotyper V 3.1 database and the mass peak lists were examined by cluster and principal component (PCA) analysis. Identification of potential biomarkers was performed using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and BLAST tool of the NCBI database. PCR identified 100% of the isolates as S. mutans. S. mutans strains were typed as serotypes c (85.6%), e (8.6%), k (4.8%), and f (0.9%). Although only the 70% of the strains tested were identified at species level by the Biotyper database, PCA and cluster analysis of mass peaks allowed the identification of 100% S. mutans isolates and its differentiation from the other oral and non-oral streptococci. One mass peak at m/z value of 9572.73 was identified as species-specific biomarker for S. mutans. No biomarkers were identified for S. mutans serotypes. KEY POINTS: • MALDI-TOF MS coupled with statistical tools for the identification of S. mutans. • Detection of species identifying biomarkers by MALDI-TOF MS. • PCR identification and serotyping of S. mutans from saliva samples.

Molecular and serological typing of Streptococcus mutans strains isolated from young Galician population: relationship with the oral health status.

The aim of this study was to determine the prevalence of Streptococcus mutans and its serotypes in samples from oral cavity of young Galician population and their relationship with the oral health state. The variables generally associated with dental caries, such as salivary flow rate, buffering capacity, eating habits, and lifestyle, were also analysed. No relationship was found between the variables studied and the presence of S. mutans in the oral cavity or the existence of dental caries. Presumptive strains of S. mutans were isolated from saliva samples from 48% of the analysed population. The use of conventional microbiological methods, API 20 Strep system, and species-specific polymerase chain reaction (PCR) allowed to substantiate the identity of the strains as S. mutans. Multiplex PCR protocols, developed in this study for the simultaneous detection of S. mutans and serotypes c, e, and f and for detection of S. mutans and serotype k, also confirmed this result and demonstrated that serotype c was predominant in the studied young Galician population (86%). Serotypes e (8%), k (3%), and f (2%) were also detected. Serotype c was detected in carious and caries-free subjects, while the remaining serotypes were only found in subjects with caries.

Diagnosis of hepatitis C virus infection in Spain: An opportunity to improve. / El diagnóstico de la infección por el virus de la hepatitis C en España: una oportunidad para mejorar.

BACKGROUND: Reflex testing of antibodies and viral load in the same sample for diagnosing hepatitis C virus infection speeds up access to treatment. However, how hepatitis C is diagnosed in Spanish hospitals is unknown. OBJECTIVE: To describe the available resources and procedures for the diagnosis of hepatitis C virus infection in Spain. METHODS: Survey sent to public and private Spanish hospitals with teaching accreditation with at least 200 beds. RESULTS: Of the 160 hospitals that met the inclusion criteria, 90 centres (response rate 56.3%) completed the survey. Two hospitals (2.2%) have no diagnostic resources, 15 (16.7%) can only test for anti-hepatitis C virus(Ab), 9 (10.0%) for Ab and viral load, 47 (52.2%) for Ab, viral load and genotype, 2 (2.2%) for Ab, viral load and core antigen, and 15 (16.7%) can perform Ab, core antigen, viral load and genotype tests. When an Ab test is positive, 28 (31.1%) hospitals perform reflex testing. When an active infection is diagnosed, some communication strategy is used in 62 (68.9%) hospitals. Approximately 44.2% of the respondents believe that all determinations needed to reach a definitive diagnosis should be done on a single blood sample. CONCLUSION: Although 81% of Spanish hospitals have the resources to perform reflex hepatitis C virus infection testing, it is only done in 31%, and less than a half of respondents believe that the definitive diagnosis should be performed on a single sample.

Prevalence and distribution of hepatitis C virus genotypes in Galicia during the period 2000-2015. / Prevalencia y distribución de los genotipos del virus de la hepatitis C en Galicia durante el periodo 2000-2015.

INTRODUCTION: We present the largest study conducted in Galicia on the prevalence and distribution of HCV genotypes/subtypes. METHODS: Retrospective study collecting the total number of patients chronically infected by HCV between 2000.01.01 to 2015.12.31 in 3of the main health areas: Santiago, Pontevedra and Vigo. RESULTS: We collected a total of 4469 patients. The median age was 50 years (IQR 57-45), 72,3% were men, 0,4% were coinfected with another genotype, 20,6% were coinfected with HIV and 35.2% with HBV. The main route of transmission was parenteral (83,1%), followed by unknown (15,3%), sexual (1,4%) and vertical (0,2%). The distribution of genotypes was: 62,9% HCV-1 (29,2% HCV-1a and 31,9% HCV-1b), 3,4% HCV-2, 21,0% HCV-3, 12,6% HCV-4 and 0,1% HCV-5. CONCLUSION: The distribution of genotypes in Galicia shows significant differences with respect to that observed in Spain. This distribution varies with age, gender, coinfection with HIV and/or HBV, and within geographical areas.

The efficiency of several one-step testing strategies for the diagnosis of hepatitis C.

BACKGROUND: implementing one-step strategies for hepatitis C diagnosis would help shorten the time to treatment access. Thus avoiding disease progression and complications, while facilitating hepatitis C virus (HCV) elimination. OBJECTIVE: to assess the validity and certainty of potential one-step strategies for the diagnosis of HCV infection and their associated cost and efficiency. METHODS: the study design is an economic appraisal of efficiency (cost/efficacy) using decision trees and deterministic sensitivity analysis. The analysis was performed from the payer perspective (Spanish National Health System), which exclusively considers the direct costs. Only the differential costs (diagnostic testing costs) were taken into account and the study was set in Spain. The efficacy of a diagnostic strategy was defined as the percentage of patients with an active HCV infection who received a positive diagnosis and the efficiency was defined as the cost per patient with a correctly diagnosed and active infection. RESULTS: the one-step strategies evaluated for the diagnosis of HCV had an acceptable validity and certainty due to the high sensitivity and specificity of the considered tests. The Ab-Ag strategy was the most efficient, followed by Ab-Ag-VL and Ab-VL. Ab-Ag was the most efficient due to the lower cost per patient tested, although the efficacy was lower than the Ab-VL efficacy. CONCLUSION: the study findings may help to establish more appropriate one-step diagnostic approaches whilst considering the efficacy and efficiency.

[Microbiological diagnosis of human immunodeficiency virus infection]. / Diagnóstico microbiológico de la infección por el virus de la inmunodeficiencia humana.

This document attempts to update the main tasks and roles of the Clinical Microbiology laboratory in HIV diagnosis and monitoring. The document is divided into three parts. The first deals with HIV diagnosis and how serological testing has changed in the last few years, aiming to improve diagnosis and to minimize missed opportunities for diagnosis. Technological improvements for HIV Viral Load are shown in the second part of the document, which also includes a detailed description of the clinical significance of low-level and very low-level viremia. Finally, the third part of the document deals with resistance to antiretroviral drugs, incorporating clinical indications for integrase and tropism testing, as well as the latest knowledge on minority variants.

[CMV DNA detection in plasma using real-time PCR based on the SYBR-Green I dye method]. / Detección de ADN de CMV en plasma mediante PCR en tiempo real utilizando SYBR-Green I como señal de amplificación.

OBJECTIVE: The aim of this study is to assess a real-time PCR technique on the LightCycler 2.0 with SYBR-Green I detection as compared to another real-time PCR method based on detection with FRET (fluorescence resonance energy transfer) probes for the quantification of CMV DNA. METHODS: The two real-time PCR methods were used to test plasma samples from immunocompromised patients with clinically suspected CMV disease, patients under follow-up without symptoms, and healthy adults. A standard curve for quantitative analysis by the SYBR-Green I method was performed with 10-fold diluted solutions of DNA from the CMV Towne strain (ATCC VR-977) cultured in MRC-5 monolayer. In addition, frozen samples from patients positive for CMV pp65 antigenemia were also analyzed and results compared using the two real time PCR methods. RESULTS: The real-time PCR technique using SYBR-Green I on the LightCycler 2.0 was a highly specific, fast, simple and reliable test to quantify CMV; moreover, it was cost-effective. CONCLUSION: Quantification of CMV DNA in plasma using this sensitive, fast, low-cost method was advantageous for the diagnosis and follow up of patients with opportunistic CMV infection, which are increasingly more frequent in our daily hospital clinical practice.

[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.

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.