Evaluation of a novel multiplex qPCR method for rapid detection and quantification of pathogens associated with calf diarrhoea.

AIMS: Diarrhoea is a common health problem in calves and a main reason for use of antimicrobials. It is associated with several bacterial, viral and parasitic pathogens, most of which are commonly present in healthy animals. Methods, which quantify the causative agents, may therefore improve confidence in associating a pathogen to the disease. This study evaluated a novel commercially available, multiplex quantitative polymerase chain reaction (qPCR) assay (Enterit4Calves) for detection and quantification of pathogens associated with calf-diarrhoea. METHODS AND RESULTS: Performance of the method was first evaluated under laboratory conditions. Then it was compared with current routine methods for detection of pathogens in faecal samples from 65 calves with diarrhoea and in 30 spiked faecal samples. The qPCR efficiencies were between 84%-103% and detection limits of 100-1000 copies of nucleic acids per sample were observed. Correct identification was obtained on 42 strains of cultured target bacteria, with only one false positive reaction from 135 nontarget bacteria. Kappa values for agreement between the novel assay and current routine methods varied between 0.38 and 0.83. CONCLUSION: The novel qPCR method showed good performance under laboratory conditions and a fair to good agreement with current routine methods when used for testing of field samples. SIGNIFICANCE AND IMPACT OF STUDY: In addition to having fair to good detection abilities, the novel qPCR method allowed quantification of pathogens. In the future, use of quantification may improve diagnosis and hence treatment of calf diarrhoea.

Extensive microbiological respiratory tract specimen characterization in critically ill COVID-19 patients.

Microbial co-infections may contribute to the pulmonary deterioration in COVID-19 patients needing intensive care treatment. The present study portrays the extent of co-infections in COVID-19 ICU patients. Conventional culture, molecular detections for atypical aetiologies, QiaStat-Dx® respiratory panel V2 detecting 21 respiratory pathogens and ribosomal DNA genes 16S/18S amplicon-based microbiome analyses were performed on respiratory samples from 34 COVID-19 patients admitted to the ICU. Potential pathogens were detected in seven patients (21%) by culturing, in four patients (12%) by microbiome analysis and in one patient (3%) by respiratory panel. Among 20 patients receiving antibiotics prior to ICU admission, fungi (3 Candida albicans, 1 C. tropicalis, 1 C. dubliniensis) were cultured in 5 (15%) endotracheal aspirates. Among 14 patients who were antibiotic-naive at ICU admission, two patients (6%) had bacterial respiratory pathogens (Staphylococcus aureus, Streptococcus pseudopneumoniae) cultured in their endotracheal aspirates. Microbiome analysis recognized four potential respiratory pathogens (3 Haemophilus influenza, 1 Fusobacterium necrophorum) isolated in samples from four other patients (12%). QiaStat-Dx® respiratory panel V2 detected adenovirus in one patient (3%). The prevalence of pulmonary microbial co-infections is modest among COVID-19 patients upon admission to ICU. Microbiome analysis complements conventional microbial diagnostics in characterization of respiratory co-infections.