A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva.

In response to the SARS-CoV-2 pandemic, we developed a multiplexed, paired-pool droplet digital PCR (MP4) screening assay. Key features of our assay are the use of minimally processed saliva, 8-sample paired pools, and reverse-transcription droplet digital PCR (RT-ddPCR) targeting the SARS-CoV-2 nucleocapsid gene. The limit of detection was determined to be 2 and 12 copies per µl for individual and pooled samples, respectively. Using the MP4 assay, we routinely processed over 1,000 samples a day with a 24-h turnaround time and over the course of 17 months, screened over 250,000 saliva samples. Modeling studies showed that the efficiency of 8-sample pools was reduced with increased viral prevalence and that this could be mitigated by using 4-sample pools. We also present a strategy for, and modeling data supporting, the creation of a third paired pool as an additional strategy to employ under high viral prevalence.

Associations between presence of Bartonella species deoxyribonucleic acid and complete blood cell count and serum biochemical changes in client-owned cats.

BACKGROUND: Infection with Bartonella species is common in cats but reported effects of bacteremia on laboratory variables differ. OBJECTIVES: Evaluate for associations between Bartonella bacteremia and CBC and serum biochemical changes in sick and healthy cats throughout the United States. ANIMALS: A total of 3964 client-owned cats. METHODS: Retrospective cohort study using submissions to a commercial laboratory between 2011 and 2017. Serum biochemistry and CBC abnormalities (categorized as above or below reference intervals), age, and location (high- or low-risk state for Ctenocephalides felis) in presumed healthy and sick cats were evaluated for associations with presence of Bartonella spp. DNA, detected by PCR. Univariate and multivariable logistic regression analyses were performed. RESULTS: Bartonella spp. DNA was amplified from 127 (3.2%) of 3964 cats; 126 (99.2%) of 127 were from high flea risk states and 121 (95.3%) of 127 were presumed sick. Fever of unknown origin was the most common PCR panel requested. In the multivariable analysis, neutrophilia, decreased ALP activity, clinical status (presumed sick), and young age (≤2 years) each were positively associated whereas neutropenia and hyperproteinemia both were negatively associated with Bartonella spp. bacteremia. Presence of Bartonella spp. DNA had no association with test results for other infectious disease agents. CONCLUSIONS AND CLINICAL IMPORTANCE: In both healthy and sick cats, active Bartonella infections had minimal association with clinically relevant laboratory abnormalities. However, based on these results, in areas considered high risk for C. felis, active infection with Bartonella spp. is a reasonable differential diagnosis for cats presented with unexplained fever and neutrophilia, particularly if the cat is young.