Inmaculada Casas

Simultaneous Detection of Fourteen Respiratory Viruses in Clinical Specimens by Two Multiplex Reverse Transcription Nested-PCR Assays

There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT‐nested PCR assay has been used widely for simultaneous detection of non‐related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT‐PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT‐PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT‐PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population. J. Med. Virol. 72:484–495, 2004.

Diagnostic system for rapid and sensitive differential detection of pathogens

Naturally emerging and deliberately released pathogens demand new detection strategies to allow early recognition and containment. We describe a diagnostic system for rapid, sensitive, multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples.

Multiple simultaneous viral infections in infants with acute respiratory tract infections in Spain

Abstract Background The clinical significance of the presence of more than one type of virus in the respiratory specimens of children with respiratory infections is not clear. Objectives To describe the clinical characteristics of multiple viral infections versus single infection by respiratory syncytial virus (RSV) in hospitalized infants. Study design This is a prospective study conducted in all infants under 2 years of age admitted for acute respiratory infection (September 2000–June 2003) in a secondary teaching hospital. Virological diagnosis was made by two different multiplex reverse transcription-nested polymerase chain reaction (RT-PCR) assays in nasopharyngeal aspirates. We describe the clinical characteristics of the patients with multiple viral infections and compare them to a group of 86 randomly selected patients infected only with RSV. Results 749 specimens taken were analyzed. Respiratory viruses were detected in 65.9% of the samples. 86 children had multiple viral infections (17.4% of all positive specimens). The most frequent clinical diagnosis in this group was recurrent wheezing in 44% and bronchiolitis in 52%. Fever was significantly more frequent (p <0.001), hospital stays were longer (p =0.05), and antibiotic treatment was used more (p =0.03) in infants with multiple viral infections than in the RSV-infected group. Conclusions Multiple viral infections are frequent in hospitalized children with respiratory tract disease (17.4%). Multiple viral infections are linked to higher fever, longer hospital stays and more frequent use of antibiotics than in the case of infants with single RSV infections.

Detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three‐year prospective study

Aim:  We have designed a study with the objective of describing the clinical impact of other viruses different from the respiratory syncytial virus (RSV) in hospitalized infants with bronchiolitis.

Ten Years of Global Evolution of the Human Respiratory Syncytial Virus BA Genotype with a 60-Nucleotide Duplication in the G Protein Gene

ABSTRACT The emergence of natural isolates of human respiratory syncytial virus group B (HRSV-B) with a 60-nucleotide (nt) duplication in the G protein gene in Buenos Aires, Argentina, in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003) and their dissemination worldwide allowed us to use the duplicated segment as a natural tag to examine in detail the evolution of HRSV during propagation in its natural host. Viruses with the duplicated segment were all clustered in a new genotype, named BA (A. Trento et al., J. Virol. 80:975-984, 2006). To obtain information about the prevalence of these viruses in Spain, we tested for the presence of the duplicated segment in positive HRSV-B clinical samples collected at the Severo Ochoa Hospital (Madrid) during 12 consecutive epidemics (1996-1997 to 2007-2008). Viruses with the 60-nt duplication were found in 61 samples, with a high prevalence relative to the rest of B genotypes in the most recent seasons. Global phylogenetic and demographic analysis of all G sequences containing the duplication, collected across five continents up until April 2009, revealed that the prevalence of the BA genotype increased gradually until 2004-2005, despite its rapid dissemination worldwide. After that date and coinciding with a bottleneck effect on the population size, a relatively new BA lineage (BA-IV) replaced all other group B viruses, suggesting further adaptation of the BA genotype to its natural host.

Prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in Spain

Human metapneumovirus (hMPV), a condition recently described in the Netherlands, causes lower respiratory infections, particularly in young children and among the elderly. The objective of this study was to describe the characteristics of hMPV infections in hospitalized infants <2 years of age and to compare them to those of infections caused by respiratory syncytial virus (RSV). A prospective study was conducted on the clinical characteristics of infants admitted to hospital for respiratory infection through 5 years. Simultaneous detection of influenza A, B, and C viruses, RSV, and adenoviruses was performed in clinical samples by multiple reverse transcription nested‐PCR assay. The presence of hMPV was tested in all samples using two separate RT‐PCR tests. Some respiratory virus was detected in 70.5% of the 1,322 children included in the study. hMPV was found in 101 of the positive nasopharyngeal aspirates (10.8%), and was the most common virus after RSV and rhinovirus. Peak incidence was found in March. Over 80% of children were <12 months. The more common diagnoses were bronchiolitis (49.5%) and recurrent wheezing (45.5%). Fifty‐four percent of cases required oxygen therapy and, one percent, assisted ventilation. Thirty percent were co‐infections, with clinical characteristics indistinguishable from single infections. Seventy‐one hMPV single infections were compared to 88 RSV single infections. hMPV infections were significantly more frequent than RSV in infants older than 6 months (P = 0.04). Recurrent wheezing was diagnosed more frequently in hMPV patients (P = 0.001). All other variables tested were similar, in both groups. hMPV was the third most frequent virus after RSV and rhinovirus in infants <2 years of age, hospitalized for respiratory infection, and was associated with bronchiolitis and recurrent wheezing. hMPV predominantly occurred in spring. Co‐infections were frequent and clinically similar to single infections and RSV infections. Pediatr Pulmonol.

High incidence of human bocavirus infection in children in Spain

Abstract Background The newly identified human bocavirus (HBoV), a member of the Parvoviridae family, has been associated to low respiratory tract infections in young children. Objectives To present the epidemiological profile and the main clinical characteristics showed by children infected with this virus in Spain. Study design We have studied the incidence of HBoV and other 15 respiratory viruses in 917 nasopharyngeal aspirates taken from 730 infants and children under age of 14 with acute lower respiratory tract infection from September-04 to August-06. Results HBoV was detected in 123 samples (13.4%) showing a seasonal distribution with November and December as the peak months. Out of the 558 samples which rendered a positive result for at least one of the virus tested, HBoV (22%) ranked fourth behind respiratory syncytial virus (181, 32%), adenoviruses (155, 28%) and rhinoviruses (136, 24%). Co-infections with HBoV and other respiratory viruses were detected in 74 out of 123 HBoV-positive specimens (60%). In addition, HBoV was also found in stool and, for the first time, in urine samples. Conclusions Results obtained provide further evidence that HBoV is involved in acute lower respiratory tract infections. HBoV-associated disease should not be limited to the respiratory tract.

Global Distribution of Novel Rhinovirus Genotype

Global surveillance for a novel rhinovirus genotype indicated its association with community outbreaks and pediatric respiratory disease in Africa, Asia, Australia, Europe, and North America. Molecular dating indicates that these viruses have been circulating for at least 250 years.

Detection of Respiratory Viruses and Subtype Identification of Influenza A Viruses by GreeneChipResp Oligonucleotide Microarray

ABSTRACT Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus.

Detection of enteroviral RNA and specific DNA of herpesviruses by multiplex genome amplification.

A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to allow rapid, sensitive and simultaneous detection of enteroviral RNA and herpesviral DNA specific sequences in a single tube. The method involves a reverse transcription step followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and specific herpesviruses DNA. Nested amplification utilises primers designed to anneal into the amplification product from the first reaction. Individual viruses were then detected and differentiated by the size of their PCR products determined using ethidium bromide stained agarose gels. To exclude false negatives due to sample inhibitors an internal amplification control, a cloned fragment of DNA from Pseudorabies virus (PRV DNA) was included in the reaction mixture. Detection levels between 0.01 and 0.001 TCID50 of prototype strains of Polio and Coxsackie type B viruses and between 1 and 100 molecules of cloned-DNA of herpesviruses prototype strains were achieved. The RT multiplex PCR method proved capable of detecting enteroviral RNA or herpesviral DNA in cerebro spinal fluid (CSF) samples from patients with aetiologically well characterized encephalitis or aseptic meningitis.