Cultures were incubated overnight at 37 °C with 5% CO2, and if no growth was present in a culture after this time, it was reincubated for another 24 hours. Researchers used standard techniques (assessment of growth in 0, 1, 2, 3, or 4 quadrants) to measure the presence of pathogenic bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Growth in 3+ and 4+ quadrants was identified as heavy growth.


Method for Testing Bacterial Culture with NP Swab

"Nucleic acid extraction was performed with the ABI MagMax96 Express automated instrument and the MagMax Viral Isolation Kit. Individual real-time reverse transcriptase (RT-PCR) assays for the detection and subtyping of adenovirus, influenza subtypes A/B/C, human metapneumovirus, human rhinovirus, parainfluenza virus subtypes 1–4, and respiratory syncytial virus were performed. All specimens were tested for RNase P to confirm RNA integrity and monitor for PCR inhibitors. Samples were run in batches on an ABI Step One Plus 96-well real-time PCR instrument. Negative and positive controls were included with each run, including RNA runoff transcripts to generate a quantitation curve. Specimens were considered positive if the Ct value was <40 cycles."


Method for Viral Detection with NP Swab

"Nucleic acid extraction was performed with the ABI MagMax96 Express automated instrument from one of the four aliquots prepared from the original sample. Gene expression of interleukin-6 (IL-6), IL-10, IL-13, IL-17, IL-25, and IL-33; interferon: α, β, γ, and λ; and IL-8 was measured using exon-spanning primers and probes according to the manufacturer’s instructions (TaqMan™, Applied Biosystems). All values were normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) housekeeping gene."

Method for Cytokine Measurement with NP Swab

"RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s protocol from sample prepared from the tip of the swab. RNA purity was assessed using Qubit 2.0 fluorometer and Agilent TapeStation 2200 for RNA quantity and quality. Host RNA-seq libraries were generated using Illumina TruSeq RNA Access, which converts total RNA into template molecules, followed by sequence-specific capture of coding RNA. The complementary DNA (cDNA) libraries were quantified using Qubit 2.0 fluorometer, and the quality was examined using Agilent Tapestation 2200.The cDNA libraries were pooled at a final concentration of 2.0 pM. Cluster generation and paired-read, dual-indexed 75 bp sequencing was performed on Illumina NextSeq 500."

Method for RNA Sequencing with NP Swab

"Total nucleic acids were extracted from a frozen specimen without a buffer using the DNeasy UltraClean DNA Extraction Kit (Qiagen) and quantified using the Qubit™ assay (Invitrogen) from one of the four aliquots prepared from the original sample. The 16S rRNA gene V4–V5 region was amplified using a modified 515F–806R primer pair.20 Sequencing of amplicons was completed using the Illumina MiSeq platform to generate 2 × 250 paired-end reads."

Method for 16S rRNA gene amplicon sequencing with NP Swab

https://media.springernature.com/full/springer-static/image/art%3A10.1038%2Fs41390-019-0498-1/MediaObjects/41390_2019_498_Fig1_HTML.png?as=webp

Fig. 1: Sample Processing after Partitioning for Multiple Testing with NP Swab

- Bacterial culture

- Viral detection (using PCR)

- Cytokine measurement (using quantitative PCR)

- RNA-sequencing 

- 16S ribosomal RNA gene sequencing

Syracuse University

In clinical research, the collection of multiple biological samples from a single patient, especially a child, is often difficult. To study the effectiveness of multiple testing with a single NP swab, one study used NP swabs to test for bacterial culture, viral detection using PCR, cytokine measurement by quantitative PCR, RNA-sequencing (RNA-seq), and 16S ribosomal RNA gene sequencing in children with diagnosed acute sinusitis (AS). 


Multiple Testing with Nasopharyngeal (NP) Swabs

Lopez, S.M.C., Martin, J.M., Johnson, M. et al. A method of processing nasopharyngeal swabs to enable multiple testing. Pediatr Res 86, 651–654 (2019). https://doi-org.libezproxy2.syr.edu/10.1038/s41390-019-0498-1
https://www.nature.com/articles/s41390-019-0498-1

A method of processing nasopharyngeal swabs to enable multiple testing

The study used NP swabs for testing in children aged 2–12 years (mean age 5.6, SD 2.7 years) with acute sinusitis, diagnosed according to American Academy of Pediatrics guidelines. Eligible subjects had either 1) 11–29 days of non-improving daytime cough and/or rhinorrhea, or (2) 6-10 days of worsening nasal symptoms, or fever after improvement period. Excluded from the sample were children 1) with severe AS 2) who received antibiotic treatment within 15 days of the study 3) with possible other infection, with immune deficiency, cystic fibrosis, ciliary dyskinesis, or major developmental delay, and with asthma or allergic rhinitis. 174 children enrolled in the study, but due to cost restrictions, RNA-sequencing was performed on only 25 children.

Sample for Study of Multiple Testing with NP Swabs

Multiple Tests that can be Performed with NP Swab

In the study of multiple testing, the NP swabs used were ESwab, with 1 ml liquid amies, and FLOQswab. The thin, flexible, sterile, flocked swab was gently inserted in one nostril, swept the nasal cavity, and brought into contact with the pharyngeal wall. Swabs were refrigerated at 2–8 °C until transported to the laboratory on ice for testing.


Method for Using NP Swab in Study of Multiple Testing

Chi-squared (χ2) test was used to compare distributions, and paired t test as well as analysis of variance were used to compare mean values.

Statistical Method For Multiple Testing with NP Swabs

As a less-invasive measure, mid-turbinate sampling is often employed as an alternative to NP swabs and is similarly sensitive to most viral and bacterial pathogens. Further research is needed to determine whether the protocol described in this study could potentially be performed using a mid-turbinate swab.

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