Viral Aetiology And Clinical Characteristics Of Community-acquired Pneumonia In Adults

Establishment of a rapid cell culture system for dignosis of common respiratory virusesBackground: There are several diagnostic techniques available for viral detection, including viral isolation, viral nucleic acid detection, viral antigen dectection and serum specific antibody. All these methods are routinely used for viral detection in some developed countries, but not available in majority of hospitals in mainland China. In limited hospitals that viral detection is available for clinical practice, the most commonly used methods are immunofluorescence, polymerase chain reaction (PCR), or virus specific IgM antibody in acute phase serum. Viral isolation, as the"gold standard"of diagnosis of viral infection, is rarely available for clinical application. Establishment of multiple modalities system, including molecular diagnosis, cell culture for viral isolation and convalescent serum antibodies, should be important for the clinical monitoring and diagnosis of viral respiratory infections.Objective: To establish a rapid cell culture system, which combined enhanced cell culture and direct IF, for detection of common respiratory viruses, and to test the sensitivity of this system.Methods: Nasopharyngeal swab or/and throat swab samples were collected from adults that admitted to Emergency Department of Guangdong Provincial Hospital of Traditional Chinese Medicine (Guangzhou, China) and from children that admitted to Shenzhen Children's Hospital (Shenzhen, China) with a diagnosis of acute respiratory infection. Samples were inoculated onto MDCK, LLC-MK2 and HEp-2 cells, followed by centrifugation at 3000rpm for 60minutes, and then equal volume of cell specific medium was added. Plates were incubated at 37℃in 5% CO2 and inspected three to four times weekly for the presence of a cytopathic effect (CPE). Hemagglutination test (HAT) was performed at 36 to 48 hours and 5 to 7 days after inoculation both for MDCK and LLC-MK2 cells. In cultures with positive CPE or HAT, direct IF was performed to identify influenza viruses type A and B (Flu A, Flu B), respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza viruses type 1,2 and 3 (PIV-1,2,3). All negative cell cultures were screened at 7 days post-inoculation by direct IF. Standard virus strains from American Type Culture Collection were inoculated onto corresponding sensitive cells, after which cells were scraped down and scattered onto multiwell slides 3 to 7 days after inoculation. Cells in slides were fixed in acetone, after which stored at 4℃and serveed as positive controls during direct IF.Results: During January and April of 2009, 88 viruses were isolated from 641 nasopharyngeal or throat swab samples at a virus isolation rate of 13.6%. The most common virus isolated was Flu B (N=39, 6.1%), followed by Flu A (N=20, 3.1%). The other viruses were PIV-3 (N=15, 2.3%), ADV (N=8, 1.2%), RSV (N=2, 0.3%), PIV-1 (N=2, 0.3%), PIV-2 (N=1, 0.2%). There was one patient coinfected by two respiratory viruses (Flu A and Flu B).Conclusion: A rapid cell culture system for diagnosis of common respiratory viruses was build up with good sensitivity and specificity. A suitable number of samples tested each time was forty-four. Turnaround time for a posivitve result varied among different viruses from 2 to 5 days. The rapid cell culture system was suitable for clinical use.Viral aetiology and clinical characteristics of community-acquired pneumonia in adultsBackground: Community-acquired pneumonia (CAP) is one of the most common infectious diseases that threaten human health. In China, pneumonia ranks fifth among all causes of death in humans. However, there are limited data concerning the aetiology of CAP, with about 17% to 48% of patients unknown. Since 20th century, several disasters in humans have been caused by global pandemics of acute respiratory viral infectious disease, including four influenza pandemics in 20th century, high pathogenic avian influenza infection in humans that was first reported in Hong Kong in 1997, severe acute respiratory syndrome (SARS) in 2003, and so on. Up until March 7, 2010, at least 16,713 individuals died since outbreak of global pandemic (H1N1) 2009 influenza, among which 796 deaths were Chinese. Trend of pandemic 2009 influenza remains unpredictable. Southern China is considered as an influenza epicenter by scholars worldwide for its special geographic location, maritime monsoon climate and type of agriculture. Moreover, SARS is also thought to be originated from Guangdong province. Additionally, Guangzhou, a central city in southern China, communicates closely with other cities in domestic and other countries abroad. Furthermore, epidemiological surveillances in recent show that viral pneumonia is more commonly reported than previously estimated. Respiratory viruses have been considered as an important pathogen causing pneumonia. Guideline of diagnosis and treatment of CAP, however, does not provide us much recommendation for evaluation and management of viral pneumonia, owing to lack of detailed information concerning the incidence and clinical features.Objective: To estimate incidence of viral pneumonia and analyze clinical characteristics of CAP with diffirent aetiology in adults.Methods: Consecutive adult patients diagnosed of CAP during April and December of 2009 were included, with history of illness recorded. Paired serologies were routinely performed by hemagglutination inhibition assay and indirect IF. Swab samples were processed for study of respiratory viruses through isolation of viruses in rapid cell culture system and detection of nucleic acids by reverse transcription polymerase chain reaction (RT-PCR) assays. Viral aetiology was considered definitive if at least one of the above tests was positive.Results:①Overall 149 CAP patients were enrolled, with 84 males. The median (interquatile range, IQR) ages were 60 (35～77) years. Swab samples were available in all patients and paired sera in 70 cases. Eighty-nine patients were accompanied by at least one underlying condition.②Viral aetiology was established in 48 cases (32.2%) of 149 patients. Incidence of viral pneumonia in patients aged 16 to 19 years (54.5%) and patients older than 79 years (57.3%) were higher than other age groups, with statistical significance (P=0.023). Forty-four patients were infected by a single virus (Flu A 24 patients, Flu B 5 patients, PIV-3 11 patients, PIV-1 and ADV 2 patients each) and four cases by two respiratory viruses. Three viral pneumonia patients had a positive bacterial culture of sputum. Among 52 virus strains, 18 strains were dectected by rapid cell culture system, 41 strains by RT-PCR and 21 strains by serological survey, respectively. Compared to rapid cell culture system in 70 subjects that double sera were available, RT-PCR was more sensitive (P=0.003), gave a specificity of 80.7%, and serological survey similar sensitive (P=0.118), gave a specificity of 75.4%.③The median (IQR) age of viral pneumonia patients was 58(30～79) years, with 27 males. Twenty-six cases had at least one underlying condition. There were 18.8% of viral pneumonia patients that had a history of exposure to similar febrile patients. Though it seemed higher than viral negative patients (9.9%), no statistical significance was found (P=0.13).④Fever≥39℃(66.7%), fatigue (64.6%), purulent sputum (52.1%), sore throat (45.8%), dysnea (41.7%) and coryza (41.7%) were the most common symptoms in viral pneumonia patients. Some Flu A or PIV-3 infected patients manifested hemoptysis and chest pain. Dyspnea and gastrointestinal symptoms were also common in Flu and PIV-3 infected patients.⑤A positive image in more than one lobe was found in 27 viral pneumonia patients (56.3%) and pleural effusion in 6 cases (12.5%). It was similar to viral negative patients (P=0.384 and P=0.371, respectively).⑥Viral pneumonia patients were more probably to show CURB-65 score≥2 and any complications than viral negative patients, though without any statistical significance (P=0.177 and P=0.063). Oxygen therapy and electrolyte disturbances in blood was also more common in viral pneumonia patients than others (54.2% vs 31.7%, P=0.008 and 37.5% vs 14.9%, P=0.002). Patients that had underlying conditions seemed more likely to develop respiratory failure and impared renal fuction than patients with no underlying conditions.Conclusion:1. Virus was one of the common pathogens of CAP. Within the limits of viruses that were studied, Flu A and PIV-3 was the most common virus.2. Sensivity of viral detection assays varied among each other. They should be chosen according to the course of illness.3. Clinical characteristics were unable to reliably distinguish viral from bacterial aetiology. It was the same in the discrimination of specified viral pneumonia.4. Bacteria co-infection in viral pneumonia patients was possible.5. There was an urgent need to consider routine laboratory detection in hospitalized CAP patients for an adequate diagnosis of respiratory viruses, especially in severe individuals.