Multiplex Urinary Antigen Detection for 13 Streptococcus pneumoniae Serotypes Improves Diagnosis of Pneumococcal Pneumonia in South African HIV-Infected Adults.

A serotype-specific urinary antigen detection (UAD) assay for 13 serotypes included in the pneumococcal conjugate vaccine (PCV13) was recently reported as a useful diagnostic tool for pneumococcal pneumonia. We aimed to assess the diagnostic accuracy of the UAD in HIV-infected South African adults. Urine specimens from a well-defined cohort of HIV-infected South African adults with pneumonia were evaluated retrospectively in the UAD assay. Pneumonia was considered pneumococcal if either sputum Gram stain, sputum culture, blood culture, or the immunochromatographic (ICT) BinaxNow S. pneumoniae test (composite diagnostic) was positive. Among 235 enrolled pneumonia patients, the UAD assay was more frequently positive (104 [44.3%]) than the composite diagnostic (71 [30.2%]; P < 0.001) and increased the pneumococcal etiology from 30.2% by an additional 22.6% to 52.8%. The UAD assay detected more pneumococcal etiologies (45.0%) than the serotype-independent ICT (23.4%, P < 0.001). UAD identified 6/7 patients with PCV13 serotype bacteremia without misclassification of bacteremia episodes due to non-PCV13 serotypes. UAD was positive for 5.1% of asymptomatic HIV-infected persons, with higher rates among those with nasopharyngeal carriage. Concordance between serotypes identified by UAD and by Quellung reaction and PCR serotyping was 70/86 (81.4%). UAD identified the dominant serotype in multiple serotype carriage. This study confirms the utility of the UAD assay for HIV-infected adults comparing favorably with other diagnostic tests. A highly valent UAD may become a new standard for detection of pneumococcal pneumonia in adults. Prior to PCV introduction, at least 53% of pneumonia cases were due to pneumococci in HIV-infected South African adults.

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens.

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.

Etiology and Factors Associated with Pneumonia in Children under 5 Years of Age in Mali: A Prospective Case-Control Study.

BACKGROUND: There are very limited data on children with pneumonia in Mali. The objective was to assess the etiology and factors associated with community-acquired pneumonia in hospitalized children <5 years of age in Mali. METHODS: A prospective hospital-based case-control study was implemented in the Pediatric department of Gabriel Touré University Hospital at Bamako, Mali, between July 2011-December 2012. Cases were children with radiologically-confirmed pneumonia; Controls were hospitalized children without respiratory features, matched for age and period. Respiratory specimens, were collected to identify 19 viruses and 5 bacteria. Whole blood was collected from cases only. Factors associated with pneumonia were assessed by multivariate logistic regression. RESULTS: Overall, 118 cases and 98 controls were analyzed; 44.1% were female, median age was 11 months. Among pneumonia cases, 30.5% were hypoxemic at admission, mortality was 4.2%. Pneumonia cases differed from the controls regarding clinical signs and symptoms but not in terms of past medical history. Multivariate analysis of nasal swab findings disclosed that S. pneumoniae (adjusted odds ratio [aOR] = 3.4, 95% confidence interval [95% CI]: 1.6-7.0), human metapneumovirus (aOR = 17.2, 95% CI: 2.0-151.4), respiratory syncytial virus [RSV] (aOR = 7.4, 95% CI: 2.3-23.3), and influenza A virus (aOR = 10.7, 95% CI: 1.0-112.2) were associated with pneumonia, independently of patient age, gender, period, and other pathogens. Distribution of S. pneumoniae and RSV differed by season with higher rates of S. pneumoniae in January-June and of RSV in July-September. Pneumococcal serotypes 1 and 5 were more frequent in pneumonia cases than in the controls (P = 0.009, and P = 0.04, respectively). CONCLUSIONS: In this non-PCV population from Mali, pneumonia in children was mainly attributed to S. pneumoniae, RSV, human metapneumovirus, and influenza A virus. Increased pneumococcal conjugate vaccine coverage in children could significantly reduce the burden of pneumonia in sub-Saharan African countries.

Genomic load from sputum samples and nasopharyngeal swabs for diagnosis of pneumococcal pneumonia in HIV-infected adults.

Quantitative lytA real-time PCR (rtPCR) results from nasopharyngeal (NP) swabs distinguish community-acquired pneumococcal pneumonia (CAP) from asymptomatic colonization. The use of an optimized cutoff value improved pneumococcal etiology determination compared to that of traditional diagnostic methods. Here, we compare the utility of lytA rtPCR from induced sputum and from NP swabs. Pneumococcus was considered the cause of CAP in HIV-infected South African adults if blood culture, induced-sputum culture or Gram stain, urine antigen test, or whole-blood lytA rtPCR revealed pneumococcus or if lytA rtPCR from NP swabs gave a result of >8,000 copies/ml. lytA rtPCR was also performed on induced sputum. Pneumococcus was detected by lytA rtPCR from sputum in 149 (67.1%) of 222 patients with available induced sputum, whereas the results of either Gram stain or culture of sputum were positive in 105 of 229 patients (45.9%; P < 0.001). The mean copy numbers from sputum were higher when the sputum cultures were positive than when the sputum cultures were negative (7.9 versus 5.6 log10 copies/ml; P < 0.001). Against the composite diagnostic standard, a cutoff value of 10,000 copies/ml for good-quality sputum lytA rtPCR had a sensitivity of 78.1% and a specificity of 80.0%. This cutoff value performed similarly to the previously identified cutoff value of 8,000 copies/ml for NP swab lytA rtPCR (area under the curve receiver operating characteristic [AUC-ROC], 80.4% for sputum of any quality versus 79.6% for NP swabs). The AUC-ROC for good-quality sputum was 83.2%. Overall, lytA rtPCR performs similarly well on induced sputum as on NP swabs for most patients but performs slightly better if good-quality sputum can be obtained. Due to the ease of specimen collection, NP swabs may be preferable for the diagnosis of pneumococcal pneumonia.

Pneumococcal colonisation density: a new marker for disease severity in HIV-infected adults with pneumonia.

OBJECTIVE: A high genomic load of Pneumococcus from blood or cerebrospinal fluid has been associated with increased mortality. We aimed to analyse whether nasopharyngeal colonisation density in HIV-infected patients with community-acquired pneumonia (CAP) is associated with markers of disease severity or poor outcome. METHODS: Quantitative lytA real-time PCR was performed on nasopharyngeal swabs in HIV-infected South African adults hospitalised for acute CAP at Chris Hani Baragwanath Hospital, Soweto, South Africa. Pneumonia aetiology was considered pneumococcal if any sputum culture or Gram stain, urinary pneumococcal C-polysaccharide-based antigen, blood culture or whole blood lytA real-time PCR revealed pneumococci. RESULTS: There was a moderate correlation between the mean nasopharyngeal colonisation densities and increasing CURB65 scores among all-cause patients with pneumonia (Spearman correlation coefficient r=0.15, p=0.06) or with the Pitt bacteraemia score among patients with pneumococcal bacteraemia (p=0.63). In patients with pneumococcal pneumonia, nasopharyngeal pneumococcal colonisation density was higher among non-survivors than survivors (7.7 vs 6.1 log10 copies/mL, respectively, p=0.02) and among those who had pneumococci identified from blood cultures and/or by whole blood lytA real-time PCR than those with non-bacteraemic pneumococcal pneumonia (6.6 vs 5.6 log10 copies/mL, p=0.03). Nasopharyngeal colonisation density correlated positively with the biomarkers procalcitonin (Spearman correlation coefficient r=0.37, p<0.0001), proadrenomedullin (r=0.39, p=0.008) and copeptin (r=0.30, p=0.01). CONCLUSIONS: In addition to its previously reported role as a diagnostic tool for pneumococcal pneumonia, quantitative nasopharyngeal colonisation density also correlates with mortality and prognostic biomarkers. It may also be useful as a severity marker for pneumococcal pneumonia in HIV-infected adults.

Multicenter case-control study protocol of pneumonia etiology in children: Global Approach to Biological Research, Infectious diseases and Epidemics in Low-income countries (GABRIEL network).

BACKGROUND: Data on the etiologies of pneumonia among children are inadequate, especially in developing countries. The principal objective is to undertake a multicenter incident case-control study of <5-year-old children hospitalized with pneumonia in developing and emerging countries, aiming to identify the causative agents involved in pneumonia while assessing individual and microbial factors associated with the risk of severe pneumonia. METHODS/DESIGN: A multicenter case-control study, based on the GABRIEL network, is ongoing. Ten study sites are located in 9 countries over 3 continents: Brazil, Cambodia, China, Haiti, India, Madagascar, Mali, Mongolia, and Paraguay. At least 1,000 incident cases and 1,000 controls will be enrolled and matched for age and date. Cases are hospitalized children <5 years with radiologically confirmed pneumonia, and the controls are children without any features suggestive of pneumonia. Respiratory specimens are collected from all enrolled subjects to identify 19 viruses and 5 bacteria. Whole blood from pneumonia cases is being tested for 3 major bacteria. S. pneumoniae-positive specimens are serotyped. Urine samples from cases only are tested for detection of antimicrobial activity. The association between procalcitonin, C-reactive protein and pathogens is being evaluated. A discovery platform will enable pathogen identification in undiagnosed samples. DISCUSSION: This multicenter study will provide descriptive results for better understanding of pathogens responsible for pneumonia among children in developing countries. The identification of determinants related to microorganisms associated with pneumonia and its severity should facilitate treatment and prevention.

Potential implication of new torque teno mini viruses in parapneumonic empyema in children.

An unexplained increase in the incidence of parapneumonic empyema (PPE) in pneumonia cases has been reported in recent years. The present study investigated the genetic and biological specifications of new isolates of torque teno mini virus (TTMV) detected in pleural effusion samples from children hospitalised for severe pneumonia with PPE. A pathogen discovery protocol was applied in undiagnosed pleural effusion samples and led to the identification of three new isolates of TTMV (TTMV-LY). Isolated TTMV-LY genomes were transfected into A549 and human embryonic kidney 293T cells and viral replication was assessed by quantitative real-time PCR and full-length genome amplification. A549 cells were further infected with released TTMV-LY virions and the induced-innate immune response was measured by multiplex immunoassays. Genetic analyses of the three TTMV-LY genomes revealed a classic genomic organisation but a weak identity (<64%) with known sequences. We demonstrated the in vitro replication of TTMV-LY in alveolar epithelial cells and the effective release of infectious viral particles. We also showed a selective production of inflammatory mediators in response to TTMV infection. This study reports the description of replicative TTMV-LY isolated from parapneumonic effusions of children hospitalised with PPE, suggesting a potential role of the virus in the pathogenesis of pneumonia.

Viral and bacterial pathogens identification in children hospitalised for severe pneumonia and parapneumonic empyema.

Pneumonia is caused by respiratory bacteria and/or viruses. Little is known if co-infections are an aggravating factor in hospitalised children with severe pneumonia. We studied the impact of respiratory pathogens on the severity of pneumonia. Between 2007 and 2009, 52 children hospitalised with a well-documented diagnosis of community-acquired pneumonia (CAP), with or without parapneumonic empyema (PPE), were enrolled in the study. The patients were classified into 2 groups: CAP + PPE (n = 28) and CAP (n = 24). The identification of respiratory viruses and bacteria in nasopharyngeal aspirates and pleural effusion samples were performed using conventional bacterial techniques and molecular assays. Using real-time multiplex PCR and antigen detection, Streptococcus pneumoniae was the main agent identified in 76% of the cases by molecular tests and BinaxNOW® in pleural fluid. A total of 8% of pleural fluid samples remained undiagnosed. In nasopharyngeal aspirates, rhinovirus, parainfluenza viruses, human metapneumovirus, and respiratory syncytial virus were detected in both CAP and CAP + PPE populations; however, the percentage of viral co-detection was significantly higher in nasopharyngeal aspirates from CAP + PPE patients (35%) compared with CAP patients (5%). In conclusion, viral co-detection was observed mainly in patients with more severe pneumonia. Molecular biology assays improved the pathogens detection in pneumonia and confirmed the S. pneumoniae detection by BinaxNOW® in pleural effusion samples. Interestingly, the main S. pneumoniae serotypes found in PPE are not the ones targeted by the heptavalent pneumococcal conjugate vaccine.

Development and validation of a commercial real-time NASBA assay for the rapid confirmation of influenza A H5N1 virus in clinical samples.

A real-time NASBA assay for the specific confirmation of influenza A H5N1 infection was developed and evaluated using proficiency panels distributed to the UK influenza network of laboratories and clinical samples received through the Chinese National Influenza Centre in Beijing. The aim of the proficiency panels was to determine the sensitivity and specificity of the assay on a range of influenza virus types and subtypes including different clades of influenza A H5 viruses. The assay was then evaluated using 19 clinical samples obtained from seven confirmed human cases of influenza A H5N1 infection in China. The assay was shown to have a level of sensitivity of 0.01 TCID50 and 10copies/µl using RNA transcripts of the A/VietNam/1194/2004 H5N1 virus. During the evaluation the assay successfully detected H5N1 viruses known to infect humans from clades 1, 2.1, 2.2 and 2.3 as well as low pathogenic H5N3 avian influenza viruses. The clinical utility of the real-time NASBA assay was proven on a range of clinical samples from patients with confirmed H5N1 infection collected during 2005 and 2006. The real-time NASBA assay was demonstrated to be sensitive and rapid allowing for same day confirmation of a H5N1 infection direct from clinical samples.