[Meningitis/Encephalitis diagnosis in ICU using Multiplex PCR system: Is it time of change?] / Diagnóstico de meningitis/encefalitis en UCI con sistema de PCR múltiple. ¿Es tiempo de cambio?

OBJECTIVE: To evaluate the clinical impact of Meningitis/Encephalitis FilmArray® panel for the diagnosis of cerebral nervous system infection and to compare the results (including time for diagnosis) with those obtained by conventional microbiological techniques. METHODS: A prospective observational study in an Intensive Care Unit of adults from a tertiary hospital was carried out. Cerebrospinal fluid from all patients was taken by lumbar puncture and assessed by the meningitis/encephalitis FilmArray® panel ME, cytochemical study, Gram, and conventional microbiological cultures. RESULTS: A total of 21 patients admitted with suspicion of Meningitis/Encephalitis. Median age of patients was 58.4 years (RIQ 38.1-67.3), median APACHE II 18 (RIQ 12-24). Median stay in ICU and median hospital stay was 4 (RIQ 2-6) and 17 days (RIQ 14-28), respectively. The overall mortality was 14.3%. A final clinical diagnosis of meningitis or encephalitis was established in 16 patients, obtaining the etiological diagnosis in 12 of them (75%). The most frequent etiology was Streptococcus pneumoniae (8 cases). FilmArray® allowed etiological diagnosis in 3 cases in which the culture had been negative, and the results led to changes in the empirical antimicrobial therapy in 7 of 16 cases (43.8%). FilmArray® yielded a global sensitivity and specificity of 100% and 90%, respectively. The median time to obtain results from the latter and conventional culture (including antibiogram) was 2.9 hours (RIQ 2.1-3.8) and 45.1 hours (RIQ 38.9-58.7), respectively. CONCLUSIONS: The Meningitis/Encephalitis FilmArray® panel was able to establish the etiologic diagnosis faster than conventional methods. Also, it achieved a better sensitivity and led to prompt targeted antimicrobial therapy.

Circulación de virus Chikungunya en el estado Aragua (Venezuela) durante el año 2014 / Circulation of Chikungunya virus in Aragua state (Venezuela) during the year 2014

El virus chikungunya (CHIKV) es un Alfavirus causante de la fiebre chikungunya (CHIKF). En Venezuela, una región desprovista de inmunidad contra CHIKV y con presencia de Aedes aegypti y Aedes albopictus, el primer caso importado fue reportado por las autoridades sanitarias en junio de 2014. Por la relevancia del hecho, se analizaron 94 muestras de pacientes febriles que acudieron a los centros de salud públicos y privados del estado Aragua entre enero y diciembre de 2014, mediante la detección de los fragmentos de los genes nsP4 (Alfavirus) y E1 (CHIKV) utilizando técnicas moleculares, como Transcripción Reversa acoplada a Reacción en Cadena de la Polimerasa (RT-PCR) y/o secuenciación nucleotídica. Los resultados indicaron positividad en 19,2 % de las muestras analizadas. Se vieron afectados pacientes con edades entre 6 y 66 años, con predominio del sexo femenino (12/18). Clínicamente, todos los pacientes positivos a CHIKV manifestaron signos y síntomas asociados a CHIKF, tales como fiebre (18/18), artralgia (18/18) y erupción (16/18), entre otros. A pesar de que la positividad puede considerarse baja con relación a lo reportado en otras comunidades, este estudio representa el primer reporte local de detección molecular de CHIKV en Venezuela (estado Aragua) durante el año 2014.

Chikungunya virus is an Alphavirus that causes chikungunya Fever (CHIKF). In Venezuela, a region devoid of immunity against CHIKV and presence of Aedes aegypti and Aedes albopictus. The first imported case was reported by health authorities in June 2014. The relevance of the fact, 94 samples of febrile patients who came to the centers of public and private health Aragua state between january and december for detection of the nsP4 (Alphavirus) and E1 (CHIKV) fragments were analyzed by molecular techniques (Reverse Transcriptase Polymerase Chain Reaction and/or nucleotide sequencing). The results showed 19.2 % of positivity by CHIKV. Clinically all CHIKV positive patients showed signs and symptoms related with CHIKF, such as fever (18/18), arthralgia (18/18) and rash (16/18), among others. Were affected patients between the ages of 6 and 66 years with a predominance of the female sex (12/18). Although the positivity may be considered low compared to those reported in other communities, this represents the first local report of molecular detection of CHIKV in Venezuela (Aragua state) during 2014.

European survey on laboratory preparedness, response and diagnostic capacity for Crimean-Congo haemorrhagic fever, 2012.

Crimean-Congo haemorrhagic fever (CCHF) is an infectious viral disease that has (re-)emerged in the last decade in south-eastern Europe, and there is a risk for further geographical expansion to western Europe. Here we report the results of a survey covering 28 countries, conducted in 2012 among the member laboratories of the European Network for Diagnostics of 'Imported' Viral Diseases (ENIVD) to assess laboratory preparedness and response capacities for CCHF. The answers of 31 laboratories of the European region regarding CCHF case definition, training necessity, biosafety, quality assurance and diagnostic tests are presented. In addition, we identified the lack of a Regional Reference Expert Laboratory in or near endemic areas. Moreover, a comprehensive review of the biosafety level suitable to the reality of endemic areas is needed. These issues are challenges that should be addressed by European public health authorities. However, all respondent laboratories have suitable diagnostic capacities for the current situation.

Viral infections of the central nervous system in Spain: a prospective study.

The aim of the study was to determine the incidence of viruses causing aseptic meningitis, meningoencephalitis, and encephalitis in Spain. This was a prospective study, in collaboration with 17 Spanish hospitals, including 581 cases (CSF from all and sera from 280): meningitis (340), meningoencephalitis (91), encephalitis (76), febrile syndrome (7), other neurological disorders (32), and 35 cases without clinical information. CSF were assayed by PCR for enterovirus (EV), herpesvirus (herpes simplex [HSV], varicella-zoster [VZV], cytomegalovirus [CMV], Epstein-Barr [EBV], and human herpes virus-6 [HHV-6]), mumps (MV), Toscana virus (TOSV), adenovirus (HAdV), lymphocytic choriomeningitis virus (LCMV), West Nile virus (WNV), and rabies. Serology was undertaken when methodology was available. Amongst meningitis cases, 57.1% were characterized; EV was the most frequent (76.8%), followed by VZV (10.3%) and HSV (3.1%; HSV-1: 1.6%; HSV-2: 1.0%, HSV non-typed: 0.5%). Cases due to CMV, EBV, HHV-6, MV, TOSV, HAdV, and LCMV were also detected. For meningoencephalitis, 40.7% of cases were diagnosed, HSV-1 (43.2%) and VZV (27.0%) being the most frequent agents, while cases associated with HSV-2, EV, CMV, MV, and LCMV were also detected. For encephalitis, 27.6% of cases were caused by HSV-1 (71.4%), VZV (19.1%), or EV (9.5%). Other positive neurological syndromes included cerebellitis (EV and HAdV), seizures (HSV), demyelinating disease (HSV-1 and HHV-6), myelopathy (VZV), and polyradiculoneuritis (HSV). No rabies or WNV cases were identified. EVs are the most frequent cause of meningitis, as is HSV for meningoencephalitis and encephalitis. A significant number of cases (42.9% meningitis, 59.3% meningoencephalitis, 72.4% encephalitis) still have no etiological diagnosis.

Use of internally controlled real-time genome amplification for detection of variola virus and other orthopoxviruses infecting humans.

Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3'-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.

Detection and identification of orthopoxviruses using a generic nested PCR followed by sequencing.

Some orthopoxviruses are considered to be potential biological weapons. After the smallpox eradication campaign ended, routine vaccination was stopped around the world. Consequently, a significant portion of the population is now completely unprotected from infection by variola virus and related orthopoxviruses. Some of the symptoms associated with non-variola infections can be similar to smallpox, causing alert and panic situations. These infections should be considered as real public health concerns, so suitable tools for their differential diagnosis are needed. This study aims to devise a simple and easy-to-perform method that is able to detect and identify any orthopoxvirus that might cause infection in humans. In addition, the similarity of the different genes in the genomes of several species of orthopoxviruses is investigated, and orthopoxvirus-universal primer pairs in the tumour necrosis factor receptor II homologue gene are designed, taking full account of nucleotide similarity. A strategy is devised for their sensitive, rapid and cost-effective detection and identification, based on a nested PCR followed by sequencing. The efficacy of the method is tested with samples sent by the European Network of Imported Viral Diseases as part of two external quality control assays. All human orthopoxviruses assayed were detected and identified.

Molecular detection of human calicivirus among Spanish children with acute gastroenteritis.

A survey was conducted among Spanish children with gastroenteritis treated in an emergency room. Reverse transcription-PCR with specimens negative for other enteric pathogens was used. The minimum incidence of human calicivirus infection was 7.7%, with Lordsdale as the predominant genotype. The clinical features and severity of calicivirus and rotavirus were similar.

Cloning and sequence of a 3.835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans.

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3'-terminal half of a gene encoding a PEX5-like protein.

Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans.

The ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in strain EL2 (Arg-) using a gene library constructed in the double autonomously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homology (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARG5,6 gene is responsible for both the acetylglutamate kinase and acetylglutamyl-phosphate reductase activities, the second and third steps of arginine biosynthesis at the mitochondria. The C. albicans ARG5,6 gene complemented the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yeast episomal plasmid using its own regulatory signals. A set of non-integrative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6 delta strain was obtained using the common URA3-blaster strategy. In addition, we generated an arg5,6 delta null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.

[The validation of a rapid diagnostic method for urinary infection in the school-age population]. / Validación de un método de diagnóstico rápido de infección urinaria en población escolar.

Validity of visual reading of a reactive strip is measured, in comparison with automatized reading and the exam of the urinary sediment, in 562 school pupils with ages between 6 and 16 years. Using urine culture as standard reference, sensibility of the leukocyte-esterase (L-E) test, nitrites and red cells was 66.7%, 33.3% and 66.7% respectively, for the visual reading; 66.7%, 33.3% and 33.3% for automatized reading, respectively; and 66.7% for leukocyturia, and 33.3% for hematuria in the urine sediment. Specificity of tests was 84.9%, 99% and 42.7% for L-E, nitrites and red cells, in visual reading; 92.5%, 100% and 80.8% for the same tests in automatized reading, and 94.5% (leukocyturia) and 86.3% (red cells) in the urinary sediment. Validity of these diagnostic methods results different depending on the population they are applied to. Even though the multi-urine test is still the most effective test in a symptomatic population and the less expensive, it would be not recommendable its use as a screening test in a school population with hidden urinary infection.