Francisco Pozo

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.

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.

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.

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.

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. J. Med. Virol. 85:554–562, 2013.

Role of Rhinovirus in Hospitalized Infants With Respiratory Tract Infections in Spain

Background: Rhinovirus is a recognized cause of common cold, proven to cause asthma exacerbations in children. In Spain, no description exists, as yet, as to the degree of burden rhinovirus infections represent among hospitalized infants. Our aim was to describe rhinovirus infections in hospitalized children, under 2 years of age, and to compare these with patients infected with respiratory syncytial virus (RSV). Patients and Methods: The prospective study was performed between September 2003 and July 2005, in children <2 years of age, admitted at the Severo Ochoa Hospital (Leganés, Madrid) with fever or respiratory tract infection and with positive rhinovirus detection in the nasopharyngeal aspirate samples. Virologic diagnosis was made by multiplex reverse transcription-polymerase chain reaction and for some virus by direct immunofluorescent assay in nasopharyngeal samples. Demographic and clinical data of those patients with rhinovirus infection were described and compared with a group of 86 patients, infected only with RSV, randomly selected from the same population. Results: We detected 85 children admitted to hospital with rhinovirus infection. Rhinovirus was the cause of 25% of all admissions, among the total of 340 under 2-year olds diagnosed with fever or respiratory tract infection. Rhinovirus was the second viral agent identified, after RSV. Clinical diagnosis was recurrent wheezing in 48.2%; bronchiolitis in 36.5%; and pneumonia in 3.5%. Fever was present in 60% of the patients. Radiologic infiltrates were found in 22.4% of the children. In 50.6% of the infants, oxygen saturation under 95% was detected, at the time of admission. Hypoxia was present in RSV-infected children more frequently (P = 0.005). Also, in this group, final diagnosis was, most frequently, bronchiolitis (P = 0.0001), and rhinovirus-infected patients were most frequently males (P = 0.004). Conclusions: Rhinovirus was detected in hospitalized infants with respiratory tract disease and was the second most common virus after RSV. In our experience, it was the second etiologic agent associated with recurrent wheezing in hospitalized children, under the age of 2 years.

Viral Diagnosis of Neurological Infection by RT Multiplex PCR: A Search for Entero- and Herpesviruses in a Prospective Study

The diagnosis of a wide range of different neurological syndromes was established by a reverse transcription multiplex PCR assay. The presence of enterovirus and herpesviruses was studied in cerebrospinal fluid samples collected prospectively from 200 patients hospitalized with neurological diseases suspected of viral infection. Positive PCR results for enterovirus and neurotropic herpesvirus (herpes simplex, HSV, and varicella zoster, VZV) were obtained among the immunocompetent patients (55/156, 35%) who presented aseptic meningitis or encephalitis. Among immunocompromised patients the yield of positive PCR results was 41% (18/44), predominantly lymphotropic herpesviruses (15/44, 34%). Cytomegalovirus (CMV) DNA was detected in patients with several clinical syndromes, including encephalitis, chronic meningitis, retinitis, ventriculitis, polyradiculomyelitis, and myeloradiculitis. Epstein‐Barr (EBV) and VZV‐specific DNA sequences were detected in patients with either encephalitis, aseptic meningitis, and chronic meningitis. Dual infections of CMV and HSV or CMV and EBV were established in two AIDS patients with encephalitis and polyradiculomyelitis, respectively. The applications of this RT multiplex PCR assay are extensive and may prove to be particularly valuable for the rapid and sensitive diagnosis of neurological diseases in both immunocompetent and immunocompromised patients. J. Med. Virol. 57:145–151, 1999.

Detection and typing of lymphotropic herpesviruses by multiplex polymerase chain reaction

A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection and typing of all human lymphotropic herpesviruses described to date, including Ebstein Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6, variants A and B (HHV6-A, HHV6-B), human herpesvirus 7 (HHV7) and human herpesvirus 8 (HHV8). Oligonucleotide primers were designed to amplify a highly conserved region within the DNA polymerase gene. Each reaction component and thermal cycling parameters were thoroughly standardized to achieve optimal specificity and sensitivity for the PCR assay, which was estimated at about 10-100 molecules for each virus. An internal control, consisting of 100 molecules of a cloned fragment of the porcine pseudorabies herpesvirus (PrV) genome, was included to detect false negative results. To assess suitability and clinical application of the multiplex PCR method, a total of 35 well-characterized specimens, including Kaposis sarcoma skin lesions, serum, cerebrospinal fluid, saliva and urine samples, were tested. Results obtained suggest this technique could be applied as a sole diagnostic tool in several clinical settings in which herpesviral infection is suspected and differential diagnosis required, avoiding the need to test specimens by separate PCR methods.

Role of rhinovirus C respiratory infections in sick and healthy children in Spain.

Background: Recently a new genogroup of human rhinovirus (HRV) has been described and named HRV-C. The relative importance of HRV-C in viral respiratory tract illnesses is unknown. Objective: We looked for HRV-C in pediatric patients with respiratory tract infections to determine the incidence of HRV-C and its role in sick and healthy children. We describe the clinical differences associated with HRV-C infections and other HRV genogroups. Patients and Methods: From January 2004 to December 2008, a prospective study was conducted in children younger than 14 years who were admitted with respiratory infection to the Pediatrics Department of the Severo Ochoa Hospital in Madrid, Spain. Specimens of nasopharyngeal aspirate were taken for virologic study with polymerase chain reaction, and clinical data were recorded. HRV specimens were genotyped. We studied the frequency of HRV-C infections, the clinical course of these patients and the differences with other HRV genogroups (HRV-A and HRV-B). Presence of HRV-C was also studied in a group of healthy children. Results: HRV was detected in 424 of 1555 episodes of illness (27.2%) and in 26 of 211 healthy children (12.3%) (P < 0.001). We amplified at random 248 of them (227 hospitalized children and 21 healthy children): 132 (53.2%) had HRV-A, 28 (11.2%) had HRV-B, and 88 (35.4%) HRV-C. HRV-C infections were associated with asthma, recurrent wheezing, and bronchiolitis but were not significantly different from the HRV-A genogroup. Nevertheless, significant clinical differences were observed between the HRV-B genogroup and the other groups: more frequent infiltrate on chest radiograph (P = 0.017), fever (P = 0.052), diagnosis of pneumonia (P = 0.01), and antibiotic treatment (P = 0.004). Conclusions: HRV-C infections were frequent in hospitalized children with respiratory diseases and were associated with asthma, recurrent wheezing, and bronchiolitis. No clinical differences were found with the HRV-A group: HRV-B group had clinical differences with both the other groups.

Human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection.

The main objective of our study was to determine the frequency of human bocavirus (HBoV) detection in asymptomatic children and to compare it with that in children hospitalized because of respiratory infection. HBoV was detected in 5% of 116 healthy children versus 17% of 908 hospitalized children. HBoV can be detected in healthy children but with a significantly lower frequency than in ill children.