Serosurvey of human metapneumovirus infection in Croatia

Aim

To assess the seroprevalence of human metapneumovirus (hMPV) in Croatia.

Methods

During 2005, a total of 137 serum specimens from Croatian patients aged from 6 days to 51 years, without respiratory symptoms, were collected at the Croatian National Institute of Public Health. The sera were examined using the indirect immunofluorescent assay.

Results

The overall hMPV seropositivity rate in the samples tested was 77.4% (106/137). The seropositivity rate increased from 18.7% in children aged between 6 months and 1 year to 100% in people older than 20 years of age. The highest proportion of titers ≥1:512 was found in children aged from 1 to 2 years.

Conclusion

Our results suggest that hMPV infection is present in Croatia, with primary infection occurring in early childhood. This is the first study that indicates the circulation of hMPV in Croatia.Human metapneumovirus (hMPV) is a newly discovered respiratory virus assigned to the Paramyxoviridae family, Pneumovirinae subfamily, Metapneumovirus genus. It was first isolated in 2001 from nasopharyngeal aspirates obtained from young children in the Netherlands (1). Sequence analysis of several isolates identified two major genetic lineages (subtypes A and B) that can be further divided into subgroups A1, A2, B1, and B2 (2). HMPV causes acute respiratory tract infections in all age groups (3,4). In hospitalized young children, hMPV infection is commonly present as bronchiolitis with or without pneumonitis (5,6), whereas bronchitis, bronchospasm, and pneumonitis are most commonly seen in elderly patients (3). Since the initial report, hMPV has been studied all over the world and it has been reported on all continents (7). Seroprevalence surveys from the Netherlands (1), Japan (8), and Israel (9) indicated that virtually all children are infected by 5-10 years of age. The aim of this study was to demonstrate the presence of hMPV infection in Croatia, by examining sera from Croatian people for specific anti-hMPV antibodies by an indirect immunofluorescent assay (IFA).     

Sequence analysis of the large polymerase (L) protein of the US strain of avian metapneumovirus indicates a close resemblance to that of the human metapneumovirus

The complete nucleotide sequence of the large polymerase (L) protein of the avian metapneumovirus subgroup C strain Colorado was determined. The L protein gene of avian pneumovirus Colorado isolate (APV-C) was 6173 nucleotides in length from the gene-start to the gene-end and encoded a polypeptide of 2005 amino acids in length. The length of the L protein of APV-C was exactly the same as that of human metapneumovirus (hMPV) and one amino acid longer than the L protein of APV subgroup A. The L protein of APV-C showed 80% amino acid identity with the L protein of hMPV, but only 64% amino acid identity with the L protein of APV-A. The nucleotide and deduced amino acid sequences were compared with the corresponding sequences of eleven other paramyxoviruses. All six domains characteristic of paramyxovirus L proteins were also observed in the L protein of APV-C. All the polymerase core motifs in domain III were conserved to nearly 100% in the metapneumoviruses. Similarly, the putative ATP-binding motif in domain VI was completely conserved among the metapneumoviruses and differed in length, by one intermediate residue, from other paramyxoviruses. Phylogenetic analysis of the different L proteins also revealed a closer relationship between APV-C and hMPV.     

Serologic Testing for Human Granulocytic Ehrlichiosis at a National Referral Center

An indirect immunofluorescence assay (IFA) was used to identify patients with antibodies reactive to the human granulocytic ehrlichiosis (HGE) agent. Serum samples collected from clinically ill individuals were submitted to the Centers for Disease Control and Prevention by physicians via state health departments from throughout the United States and tested against a panel of ehrlichial and rickettsial pathogens. Antibodies reactive to the HGE agent were detected in 142 (8.9%) of 1,602 individuals tested. There were 19 confirmed and 59 probable (n = 78) cases of HGE as defined by seroconversion or a fourfold or higher titer to the HGE agent than to the Ehrlichia chaffeensis antigens. The average age of patients with HGE was 57 years, and males accounted for 53 (68%) of the patients. Cases of HGE occurred in 21 states; 47 (60%) of the cases occurred in Connecticut (n = 14), New York (n = 18), and Wisconsin (n = 15). Onset of HGE was identified from April through December, with cases peaking in June and July. The earliest confirmed cases of HGE occurred in 1987 in Wisconsin and 1988 in Florida. No fatalities were reported among the 78 patients with confirmed or probable HGE. Reactivity to the HGE agent and to either Coxiella burnetii, Rickettsia rickettsii, or Rickettsia typhi was infrequent; however, 74 (52%) of the 142 individuals who were positive for HGE had at least one serum sample that also reacted to the E. chaffeensis antigen. Thirty-four persons with confirmed or probable human monocytic ehrlichiosis due to E. chaffeensis also had antibodies to the HGE agent in at least one serum sample. The specific etiologic agent for 30 patients was not ascribed because of similarity of titers to both ehrlichial antigens. The use of both antigens may be required to correctly diagnose most cases of human ehrlichiosis, especially in geographic regions where both the HGE agent and E. chaffeensis occur.     

小鼠人偏肺病毒感染模型的建立

目的 建立人偏肺病毒(hMPV)感染小鼠模型,了解病毒肺内复制规律及所致病理改变,为hMPV感染免疫病理机制研究及新型防治手段开发奠定基础.方法 BALB/c小鼠经滴鼻感染荧光标记的重组hMPV,于感染后1、3、5、7、9、16 d处死小鼠并无菌获取肺组织用于病毒分离和病理检查,改良噬斑形成法检测hMPV滴度,RT-PCR法检测hMPV mRNA表达.结果 小鼠滴鼻感染hMPV后肺组织分离到病毒;肺组织病毒滴度在感染后5 d达到高峰(5.16±1.09)×105PFU/g,感染后第9大仍能检测到病毒(2.79±1.22)×102PFU/g;感染后16 d肺组织仍可检测到hMPV mRNA;病理改变在感染后3～7 d最明显,为典型的间质性肺炎改变.结论 hMPV感染BALB/c小鼠模型建立成功,可用于hMPV感染的免疫病理机制研究.     

Prospective study of human metapneumovirus infection in children less than 3 years of age

Most lower respiratory tract infections (LRTIs) in children under the age of 3 years are due to respiratory syncytial virus (RSV). Epidemiological, host, and viral factors eventually account for the severity of LRTIs, but they do not completely explain it. Human metapneumovirus (hMPV) was recently identified in children with LRTIs. In a population-based prospective multicenter study (the PRI.DE study, conducted in Germany over 2 years), we tested 3,369 nasopharyngeal secretions from children younger than 3 years of age with LRTIs for RSV A and B, influenza viruses (IVs) A and B, and parainfluenza viruses (PIVs) 1 to 3. Of the children requiring intensive care (n = 85), 18% had hMPV infections, and 60% of these children were infected with hMPV in combination with RSV. We did not detect hMPV in a randomly selected subset of RSV-positive nasopharyngeal secretions (n = 120) from children not requiring intensive care support. hMPV was detected in <1% of virus-negative samples from patients without intensive care support (n = 620). Our data support the hypothesis that coinfections with RSV and hMPV are more severe than infections with either RSV or hMPV alone, at least in children younger than 3 years of age.     

Characterization of human metapneumovirus infection of myeloid dendritic cells

Ebola virus is highly cytopathic through mechanisms that are largely unknown. We present evidence that progressive acidification of the extracellular milieu by Ebola virus-infected cells combined with reduced levels of natural cysteine protease inhibitor makes the cells vulnerable to uncontrolled proteolysis of extracellular matrix components by released active endosomal cathepsins, thereby exacerbating Ebola virus-induced cell destruction. The cell surface microenvironment was shown to be crucial in aiding this activity. Blocking the proteolytic activity with the cathepsin inhibitor E64 resulted in remarkable improvements with respect to viral cytopathicity and cell survival despite an overwhelmingly high viral load. We propose that the observed enzymatic matrix degradation, enhanced by an associated protease/inhibitor imbalance and metabolic acidosis, represents an effective viral strategy to boost infection and underlies, in part, the remarkable pathogenesis caused by Ebola virus. Further in vitro and in vivo research will establish whether a cellular protease with hemorrhagic activity is the leading cause of vascular leakage-the hallmark of Ebola virus hemorrhagic fever-and help understand the Ebola virus caused cell death.     

Acute respiratory infection by human metapneumovirus in children in southern Brazil.

BACKGROUND: Human metapneumovirus (hMPV) has been described as an etiologic agent of acute respiratory infections (ARI), mainly in pediatric patients. Viral isolation is difficult and has low sensitivity, and consequently RT-PCR assays are currently used for detection. OBJECTIVES: Detect hMPV in ARI in hospitalized children in Southern Brazil; standardize a RT-PCR for routine hMPV diagnosis; validate a positive control for molecular tests; and perform phylogenetics analyses. STUDY DESIGN: Nasopharyngeal aspirates (NPA) from 156 hospitalized children were studied. A conserved region of the nucleoprotein gene was cloned, characterized and used to standardize an RT-PCR assay. Phylogenetic analyses were performed. Clinical data were obtained from medical records. RESULTS: hMPV was detected in 6.4% of the samples. Dyspnea and wheezing were frequently reported symptoms and the most common diagnoses were bronchiolitis, acute respiratory insufficiency or laryngotracheobronchitis. Nucleotide sequence alignment revealed 97.7% identity with genotype A1 of hMPV. The detection limit of hMPV genomes by RT-PCR in clinical samples was 180 copies/microL. CONCLUSION: This is the first report of the detection and genetic characterization of hMPV infections in children with lower ARI in Southern Brazil.     

Detection and pathogenicity of human metapneumovirus respiratory infection in pediatric Italian patients during a winter--spring season.

BACKGROUND: Some diagnostic, epidemiological and clinical features of the recently discovered human metapneumovirus remain to be investigated. OBJECTIVES: To study the best approach for the diagnosis of human metapneumovirus infections by both conventional and molecular methods, along with the human metapneumovirus circulation rate in northern Italy and the severity of human metapneumovirus respiratory infections in a pediatric patient population. STUDY DESIGN: Nasopharyngeal aspirates (NPA) were taken from 306 pediatric patients during the winter-spring season 2003-2004, and examined for conventional respiratory viruses by direct fluorescent staining and cell culture, while human coronavirus and human metapneumovirus were sought by RT-PCR. RESULTS: RT-PCR detected human metapneumovirus in 40/306 (13.1%) children positive for respiratory viruses, with an incidence intermediate between that of respiratory syncytial virus (58 patients, 18.9%) and that of influenzavirus infections (29 patients, 9.5%). Phylogenetic analysis showed cocirculation of both human metapneumovirus types (A and B) as well as their relevant subtypes (A1-A2 and B1-B2). Clinically, human metapneumovirus was found to be second to human respiratory syncytial virus alone, as a cause of respiratory tract infections, while duration of virus excretion appeared to correlate with severity of infection, and virus load in NPA with the stage of respiratory infection. CONCLUSION: (i) Human metapneumovirus is a major viral pathogen in the Italian pediatric patient population; (ii) the severity of lower respiratory tract infections approaches that of human respiratory syncytial virus; (iii) there are preliminary indications that the duration of virus excretion may reach 2-3 weeks and that the level of viral load in NPA correlates with the clinical stage of human metapneumovirus infection.     

Focus on: SUNY Health Center at Brooklyn Scientific & Medical Instrumentation Center.

The Scientific and Medical Instrumentation Center (SMIC) is the clinical engineering program serving the State University of New York's Health Science Center at Brooklyn. SMIC is a separate department within the center's 354-bed University Hospital, and provides many instrumentation support services for the hospital and the center's Basic Sciences Division. Now in its 24th year, SMIC developed the nation's first mandatory initial checkout program for patient care equipment, and in 1973 published the results of a funded pilot preventive maintenance program; this served as a model for the start-up of other PM programs in hospitals across the country and overseas. Today, this 35-person department is primarily responsible for some 7,000 units used in over 60 University Hospital departments and clinics. With its interdisciplinary expertise, SMIC also provides the hospital with many other instrumentation services, including prepurchase evaluation and review, and on-site emergency instrumentation service. SMIC also develops unique devices and instruments for the center's researchers, from the prototype stage through to final construction, and may modify instruments for increased safety and efficacy.     

Seroepidemiological study of human metapneumovirus in New Delhi, India

Purpose: There are a few seroepidemiological studies reported on human metapneumovirus (hMPV) as hMPV was only discovered in the year 2001. This respiratory virus has been reported to be ubiquitous and associated with respiratory tract infections in all age groups. The present study aimed at determining the prevalence of antibodies to hMPV in children and adults of 1 month to 55 years of age. Materials and Methods: Serum samples from 100 study subjects were tested for hMPV antibody by an in-house ELISA system that used hMPV-infected cell lysate antigen. Result: The prevalence of antibody to hMPV was lowest in children less than 5 years of age (60%) and increased throughout age to > 80%. Similarly, geometric mean titres were 1:180 in children less than 5 years of age and reached a peak of 1:419 in adults over 35 years of age. Conclusion: The results show that hMPV infection is acquired early in life and re-infection in later life may maintain the seroprevalence and antibody levels in adult population.     

Surgical Service Nosocomial Infections at a Veterans Administration Medical Center

Nosocomial surgical infections at a Veterans Administration medical center were monitored from February 1986 to June 1987 (17 months). Three hundred twenty-four patients had 508 nosocomial infections, including 66 patients in whom cultures were not performed. The remaining 258 patients had 584 microorganisms, including gram-negative bacilli (56.8 percent), gram-positive cocci (33.6 percent), and yeasts (8.4 percent). The most common isolates were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, group D enterococci, and Staphylococcus epidermidis. Antimicrobial susceptibilities were similar for nosocomial and all hospital isolates combined 84.5 percent of the time; all hospital isolates combined were more resistant in 5.5 percent and nosocomial isolates in 10.0 percent. The infection rate for surgical wounds was only 2.5 percent. When all nosocomial infection sites were compared, however, the rate for lower respiratory tract and some other infections appeared high and disproportionate. The significance of this finding is questionable because of a lack of comparable published reports from other Veterans Administration medical centers. The importance of patient population at risk of obtaining some nosocomial infections (especially lower respiratory) may be greater in adult men than in other patient populations.     

Seasonality and clinical features of human metapneumovirus infection in children in Northern Alberta

Human metapneumovirus (hMPV) causes respiratory tract infections in all age groups. The characteristics of pediatric hMPV infection in Northern Alberta have not been studied. The objectives of this study were to determine the seasonality of pediatric hMPV infections over a 13-month period, the genetic relationship of hMPV isolates to hMPV detected in other parts of Canada, and the burden of illness and possible risk factors for pediatric hMPV hospitalization. Detection of hMPV by polymerase chain reaction was performed on nasopharyngeal specimens collected from outpatients and inpatients at the Stollery Children's Hospital in Edmonton, Alberta, November 12, 2002-December 31, 2003. Forty-two of 1,079 specimens were positive for hMPV (3.9%) from 41 patients (14 outpatients and 27 inpatients), with a peak incidence during January-April, but isolates were detected 10 months of the year. Co-infection was not detected in 39 specimens from which RSV had been detected. Two hMPV genetic clusters were detected, and the isolates were homologous to those of previous Canadian isolates. Four of the 14 outpatients had reactive airways disease. Possible risk factors in the 27 inpatients included prematurity (n = 8), congenital heart disease (n = 6), gastroesophageal reflux disease or aspiration (n = 6), global developmental delay (n = 5), and multiple congenital anomalies (n = 4). Risk factors for hospitalization appear to be similar to risk factors for respiratory syncytial virus hospitalization.     

Analysis of the genomic sequence of a human metapneumovirus

We recently described the isolation of a novel paramyxovirus from children with respiratory tract disease in The Netherlands. Based on biological properties and limited sequence information the virus was provisionally classified as the first nonavian member of the Metapneumovirus genus and named human metapneumovirus (hMPV). This report describes the analysis of the sequences of all hMPV open reading frames (ORFs) and intergenic sequences as well as partial sequences of the genomic termini. The overall percentage of amino acid sequence identity between APV and hMPV N, P, M, F, M2-1, M2-2, and L ORFs was 56 to 88%. Some nucleotide sequence identity was also found between the noncoding regions of the APV and hMPV genomes. Although no discernible amino acid sequence identity was found between two of the ORFs of hMPV and ORFs of other paramyxoviruses, the amino acid content, hydrophilicity profiles, and location of these ORFs in the viral genome suggest that they represent SH and G proteins. The high percentage of sequence identity between APV and hMPV, their similar genomic organization (3'-N-P-M-F-M2-SH-G-L-5'), and phylogenetic analyses provide evidence for the proposed classification of hMPV as the first mammalian metapneumovirus.     

Referral for minor mental illness: a qualitative study.

BACKGROUND: Mild depression and anxiety are common problems in general practice. They can be managed by the general practitioner (GP) alone or referred. Previous quantitative studies have shown a large variation between GPs in terms of referral behaviour. The reasons for this variation are not fully understood. AIM: To describe and analyse GP's decision-making processes when considering who should be treating patients with minor mental illness, using a qualitative method. DESIGN OF STUDY: A qualitative interview study. SETTING: Twenty-three GPs in east London and Essex. METHOD: Subjects were chosen using a purposive sampling strategy and participated in one-to-one semi-structured interviews. A grounded theory approach was used for analysis. RESULTS: Two distinct referral strategies were identified--the 'containment' and the 'conduit' approaches. In addition, referrals were found to be of two types--proactive 'referrals to' and reactive 'referrals away'; for minor mental illness the 'referrals away' were found to predominate. Emotive as well as rational responses informed GP decision making on referral. CONCLUSIONS: Explanations of the variation in referral rates need to recognise the emotive responses of individual GPs to minor mental illness. The contribution of guidelines, which assume consistently rational responses to illness, may therefore be limited.