A method of processing nasopharyngeal swabs to enable multiple testing

Abstract

Objective To develop a method to perform multiple tests on single nasopharyngeal (NP) swab. Study design We collected a NP swab on children aged 2–12 years with acute sinusitis and processed it for bacterial culture, viruses, cytokine expression, and 16S ribosomal RNA gene sequencing analysis. During the course of the study, we expand the scope of evaluation to include RNA sequencing, which we accomplished by cutting the tip of the swab. Results Of the 174 children enrolled, 126 (72.4%) had a positive bacterial culture and 121(69.5%) tested positive for a virus. Cytokine measurement, as judged by the adequate levels of a housekeeping enzyme (GAPDH), appeared successful. From the samples used for 16S ribosomal sequencing we recovered, on average, 16,000 sequences per sample, accounting for a total of 2,646 operational taxonomic units across all samples sequenced. Samples used for RNA sequencing had a mean RNA Integrity number of 6.0. Cutting the tip of the swab did not affect the recovery yield for viruses or bacteria, nor did it affect species richness in microbiome analysis. Conclusion We describe a minimally invasive sample collection protocol that allows for multiple diagnostic and research investigations in young children.

Seroconversion and Skin Mucosal Parameters during Koi Herpesvirus Shedding in Common Carp, Cyprinus carpio

“…As expected, Taqman qPCR Ct values obtained from non-invasive skin swabs were higher than those typically measured from skin samples (i.e., actb1 Ct average in skin swabs was 25.4 ± 2.1 vs. 15.6 ± 0.7 in skin tissues [ 34 ]). Ct values obtained in this study for the housekeeping gene were similar to those reported previously from buccal swab samples [ 46 ], however, the Taqman qPCR detection of the targeted genes failed in a proportion of the swabs analysed. While skin swabs might not be suitable when analysing low-level gene expression, for which mRNA enrichment or further RNA purification would be required, the non-lethal sampling method allowed for a considerable reduction in the number of animals used and allowed a close follow up of the response of each animal to the viral infection over time.…”

Pediatric Chronic Rhinosinusitis: Unmet Needs

“…Microbiome identification has occurred in the lungs of pediatric cystic fibrosis [23], in in asthma [24] and phenotypic pediatric asthma [25], the nasopharyngeal space in infants with cystic fibrosis [26], the nasal space in normal children and infants [27,28], in adults with CRS [19,20,29,30], and limited information in adults and children with allergic rhinitis [31]. The gut microbiome plays an active role in respiratory homeostatis [32], as does the microbiome balance of nasal and lower airways in asthmatic children with exacerbations [33].…”

“…Pediatric Otolaryngolgical specialists will need to be at the vanguard, coupled with new and easier capture technology [28] to provide preliminary microbiomic data in pediatric CRS. Details of antibiotic used prior to surgery and duration off antibiotic before surgery would be valuable companion data.…”

“…Several studies point the way for investigators to use the available ambulatory population of pediatric CRS patients for clinical research. The methodology of rapid nasopharyngeal testing has been established [28], and a publication from 2019 reported on thirty-one children with acute sinusitis [38]. A population of 237 children were followed for a year.…”

High-throughput SARS-CoV-2 and host genome sequencing from single nasopharyngeal swabs

“…Although our initial swab collection did not reveal any viral co-infections, especially as the current pandemic enters the regular flu and cold season, our method allows for acceleration of metagenomics analysis. 9,10 Further, advances in low pass genome calling allow the same nasopharyngeal swab to be used to gather a wealth of human genomics data and in many cases yielded enough DNA for deep sequencing of HLA type, which is a critical component of the host immunomic response. 2,3 Future infectious disease outbreaks will inevitably occur, and the strategy we describe is applicable for collection of host and viral genomic information from any respiratory virus in laboratories around the world.…”