Improving the quality and workflow of bacterial genome sequencing and analysis: paving the way for a Switzerland-wide molecular epidemiological surveillance platform.

Facing multidrug resistant (MDR) bacterial pathogens is one of the most important challenges for our society. The spread of highly virulent and resistant pathogens can be described using molecular typing technologies; in particular, whole genome sequencing (WGS) data can be used for molecular typing purposes with high resolution. WGS data analysis can explain the spatiotemporal patterns of pathogen transmission. However, the transmission between compartments (human, animal, food, environment) is very complex. Interoperable and curated metadata are a key requirement for fully understanding this complexity. In addition, high quality sequence data are a key element between centres using WGS data for diagnostic and epidemiological applications. We aim to describe steps to improve WGS data analysis and to implement a molecular surveillance platform allowing integration of high resolution WGS typing data and epidemiological data.

Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.

Non-invasive assessment of upper and lower airway infection and inflammation in CF patients.

BACKGROUND: The upper (UAW) and lower (LAW) airways of patients with cystic fibrosis (CF) have the same ion-channel defects, but little is known about similarities and differences in host immunological responses at the two levels. AIM: Identification and comparison of both levels' pathogen colonization and resulting immunological host responses. METHODS: The UAW and LAW of 40 CF patients were non-invasively assessed by nasal lavage and induced sputum. Pathogen colonization, cytology, and the concentrations of inflammatory mediators (TNF-α, MPO, matrix metalloprotease (MMP)-9, tissue inhibitor of metalloprotease (TIMP)-1, regulated upon activation, normal T-cell expressed and presumably secreted (RANTES), and interleukin (IL)-1ß, -5, -6, -8, and -10) were measured. RESULTS: Inflammatory responses were more pronounced in the LAW than the UAW. Pseudomonas aeruginosa LAW colonization is accompanied by a significantly enhanced neutrophil (PMN)-dominated response (P = 0.041) and IL-8 concentration (P = 0.01) not observed in P. aeruginosa UAW colonization. In contrast, sinonasal P. aeruginosa colonization resulted in elevated RANTES (P = 0.039) and reduced MMP-9 (P = 0.023) and TIMP-1 (P = 0.035) concentrations. Interestingly, LAW P. aeruginosa colonization was associated with reduced sinonasal concentrations of MMP-9 (P = 0.01) and TIMP-1 (P = 0.02), a finding independent of UAW colonization for MMP-9. CONCLUSION: CF UAW and LAW show distinct inflammatory profiles and differentiated responses upon P. aeruginosa colonization. Assessment of UAW colonization and MMP-9 are predictive of chronic pulmonary colonization with P. aeruginosa. Thus, this linkage between CF UAW and LAW can provide new clinical and scientific implications.