Detection of 11 Common Viral and Bacterial Pathogens Causing Community-Acquired Pneumonia or Sepsis in Asymptomatic Patients by Using a Multiplex Reverse Transcription-PCR Assay with Manual (Enzyme Hybridization) or Automated (Electronic Microarray) Detection

Abstract

Community-acquired pneumonia (CAP) and sepsis are important causes of morbidity and mortality. We describe the development of two molecular assays for the detection of 11 common viral and bacterial agents of CAP and sepsis: influenza virus A, influenza virus B, respiratory syncytial virus A (RSV A), RSV B, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Legionella micdadei, Bordetella pertussis, Staphylococcus aureus, and Streptococcus pneumoniae. Further, we report the prevalence of carriage of these pathogens in respiratory, skin, and serum specimens from 243 asymptomatic children and adults. The detection of pathogens was done using both a manual enzyme hybridization assay and an automated electronic microarray following reverse transcription and PCR amplification. The analytical sensitivities ranged between 0.01 and 100 50% tissue culture infective doses, cells, or CFU per ml for both detection methods. Analytical specificity testing demonstrated no significant cross-reactivity among 19 other common respiratory organisms. One hundred spiked "surrogate" clinical specimens were all correctly identified with 100% specificity (95% confidence interval, 100%). Overall, 28 (21.7%) of 129 nasopharyngeal specimens, 11 of 100 skin specimens, and 2 of 100 serum specimens from asymptomatic subjects tested positive for one or more pathogens, with S. pneumoniae and S. aureus giving 89% of the positive results. Our data suggest that asymptomatic carriage makes the use of molecular assays problematic for the detection of S. pneumoniae or S. aureus in upper respiratory tract secretions; however, the specimens tested showed virtually no carriage of the other nine viral and bacterial pathogens, and the detection of these pathogens should not be a significant diagnostic problem. In addition, slightly less sensitive molecular assays may have better correlation with clinical disease in the case of CAP.

Viral infection in community-acquired pneumonia: a systematic review and meta-analysis

“…Furthermore, even when viruses are detected by lower respiratory tract sampling, contamination from the upper airways may have occurred. Conversely, a study using PCR found that asymptomatic nasopharyngeal carriage in adults was common for Staphylococcus aureus (9.1%) and Streptococcus pneumoniae (6.8%), but absent for the four viruses tested: influenza virus A, influenza virus B, respiratory syncytial virus A and respiratory syncytial virus B [43].…”

Infantile respiratory syncytial virus and human rhinovirus infections: respective role in inception and persistence of wheezing

“…RSV is ubiquitous and nearly all children are infected by 2 years of age or following two RSV seasons, and LRTIs due to RSV are a leading cause of hospitalisation during the first year of life [7,[22][23][24]. However, only a subset of the infected children develops severe disease [8,[23][24][25][26][27]. In addition to the variability in severity among different RSV seasons, a number of host-related characteristics may increase the risk of hospital admission rates for RSV-induced LRTI in young children [8,[23][24][25][26][27][28].…”

“…Strictly speaking, the high sensitivity of these tests may complicate clinical interpretation, as the presence of small amounts of viral targets may not necessarily prove their pathogenetic role. However, prospective case-control studies in asymptomatic and symptomatic young children have shown that a positive RSV test result is almost always of clinical relevance, independent of viral quantity [24,25].…”

“…RSV epidemics are seasonal, with peak infection occurring during late autumn/winter to early spring in the northern hemisphere [8,[23][24][25][26][27][28]. RSV is ubiquitous and nearly all children are infected by 2 years of age or following two RSV seasons, and LRTIs due to RSV are a leading cause of hospitalisation during the first year of life [7,[22][23][24].…”

Assessment of the Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of <i>Mycoplasma pneumoniae</i> in Pediatric Community-Acquired Pneumonia

“…Daxboeck et al (8) stated that transient asymptomatic carriage of M. pneumoniae resulted from the persistence of the pathogen after previous disease and from infections during incubation periods. Asymptomatic healthy children act as carriers of M. pneumoniae at a rate of 0z-2.2z (12)(13)(14); however, because the detection of M. pneumoniae DNA in throat swabs is not necessarily indicative of a causative role of the pneumonic pathogen, an infectious state is often difficult to distinguish from carriage during the acute phase. Given the high positive predictive value of the LAMP assay in this study, the use of antimycoplasma therapy in LAMP-positive cases diagnosed with pneumonia may be reasonable.…”