The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

Acute bacterial meningitis is a medical emergency, and delays in initiating effective antimicrobial therapy result in increased morbidity and mortality. Culture-based methods, thus far considered the “gold standard” for identifying bacterial microorganisms, require 24 to 48 h to provide a diagnosis. In addition, antimicrobial therapy is often started prior to clinical sample collection, thereby decreasing the probability of confirming the bacterial pathogen by culture-based methods. To enable a fast and accurate detection of the most important bacterial pathogens causing meningitis, namely, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Streptococcus agalactiae, and Listeria monocytogenes, we evaluated a commercially available multiplex LightMix real-time PCR (RT-PCR) in 220 cerebrospinal fluid (CSF) specimens. The majority of CSF samples were collected by lumbar puncture, but we also included some CSF samples from patients with symptoms of meningitis from the neurology department that were recovered from shunts. CSF samples were analyzed by multiplex RT-PCR enabling a first diagnosis within a few hours after sample arrival at our institute. In contrast, bacterial identification took between 24 and 48 h by culture. Overall, a high agreement of bacterial identification between culture and multiplex RT-PCR was observed (99%). Moreover, multiplex RT-PCR enabled the detection of pathogens, S. pneumoniae (n = 2), S. agalactiae (n = 1), and N. meningitidis (n = 1), in four culture-negative samples. As a complement to classical bacteriological CSF culture, the LightMix RT-PCR assay proved to be valuable by improving the rapidity and accuracy of the diagnosis of bacterial meningitis.

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.

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