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Procedures for collection of induced sputum specimens from children.

ABSTRACT: In most settings, sputum is not routinely collected for microbiological diagnosis from children with lower respiratory disease. To evaluate whether it is feasible and diagnostically useful to collect sputum in the Pneumonia Etiology Research for Child Health (PERCH) study, we reviewed the literature on induced sputum procedures. Protocols for induced sputum in children were collated from published reports and experts on respiratory disease and reviewed by an external advisory group for recommendation in the PERCH study. The advisory group compared 6 protocols: 4 followed a nebulization technique using hypertonic saline, and 2 followed a chest or abdomen massage technique. Grading systems for specimen quality were evaluated. Collecting sputum from children with lower respiratory tract illness is feasible and is performed around the world. An external advisory group recommended that sputum be collected from children hospitalized with severe and very severe pneumonia who participate in the PERCH study provided no contraindications exist. PERCH selected the nebulization technique using hypertonic saline.

SUBMITTER: Grant LR

PROVIDER: S-EPMC3297553 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Procedures for collection of induced sputum specimens from children.

Grant Lindsay R LR Hammitt Laura L LL Murdoch David R DR O'Brien Katherine L KL Scott J Anthony JA

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 20120401

In most settings, sputum is not routinely collected for microbiological diagnosis from children with lower respiratory disease. To evaluate whether it is feasible and diagnostically useful to collect sputum in the Pneumonia Etiology Research for Child Health (PERCH) study, we reviewed the literature on induced sputum procedures. Protocols for induced sputum in children were collated from published reports and experts on respiratory disease and reviewed by an external advisory group for recommend ...[more]

PMID: 22403228

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Project description:BackgroundCareful consideration of experimental artefacts is required in order to successfully apply high-throughput 16S ribosomal ribonucleic acid (rRNA) gene sequencing technology. Here we introduce experimental design, quality control and "denoising" approaches for sequencing low biomass specimens.ResultsWe found that bacterial biomass is a key driver of 16S rRNA gene sequencing profiles generated from bacterial mock communities and that the use of different deoxyribonucleic acid (DNA) extraction methods [DSP Virus/Pathogen Mini Kit® (Kit-QS) and ZymoBIOMICS DNA Miniprep Kit (Kit-ZB)] and storage buffers [PrimeStore® Molecular Transport medium (Primestore) and Skim-milk, Tryptone, Glucose and Glycerol (STGG)] further influence these profiles. Kit-QS better represented hard-to-lyse bacteria from bacterial mock communities compared to Kit-ZB. Primestore storage buffer yielded lower levels of background operational taxonomic units (OTUs) from low biomass bacterial mock community controls compared to STGG. In addition to bacterial mock community controls, we used technical repeats (nasopharyngeal and induced sputum processed in duplicate, triplicate or quadruplicate) to further evaluate the effect of specimen biomass and participant age at specimen collection on resultant sequencing profiles. We observed a positive correlation (r = 0.16) between specimen biomass and participant age at specimen collection: low biomass technical repeats (represented by < 500 16S rRNA gene copies/μl) were primarily collected at < 14 days of age. We found that low biomass technical repeats also produced higher alpha diversities (r = - 0.28); 16S rRNA gene profiles similar to no template controls (Primestore); and reduced sequencing reproducibility. Finally, we show that the use of statistical tools for in silico contaminant identification, as implemented through the decontam package in R, provides better representations of indigenous bacteria following decontamination.ConclusionsWe provide insight into experimental design, quality control steps and "denoising" approaches for 16S rRNA gene high-throughput sequencing of low biomass specimens. We highlight the need for careful assessment of DNA extraction methods and storage buffers; sequence quality and reproducibility; and in silico identification of contaminant profiles in order to avoid spurious results.

Project description:The gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection diagnosis is reverse transcription (RT)-PCR from a nasopharyngeal swab specimen (NPS). Its collection involves close contact between patients and health care workers, requiring a significant amount of workforce and putting them at risk of infection. We evaluated self-collection of alternative specimens and compared their sensitivity and cycle threshold (CT) values to those of NPS. We visited acute coronavirus disease 2019 (COVID-19) outpatients to collect concomitant NPS and gargle specimens and had patients self-collect gargle and either sputum or spit specimens the next morning. We included 40 patients and collected 40 concomitant NPS and gargle specimens, as well as 40 gargle, 22 spit, and 16 sputum specimens the next day (2 patients could not produce sputum). All specimens were as sensitive as NPS. Gargle specimens had a sensitivity of 0.97 (95% confidence interval [CI], 0.92 to 1.00), whether collected concomitantly with NPS or the next morning. Next-morning spit and sputum specimens showed sensitivities of 1.00 (95% CI, 1.00 to 1.00) and 0.94 (95% CI, 0.87 to 1.00]), respectively. The gargle specimens had significantly higher mean CT values of 29.89 (standard deviation [SD], 4.63; P < 0.001) and 29.25 (SD, 3.99; P < 0.001) when collected concomitantly and the next morning, respectively, compared to NPS (22.07 [SD, 4.63]). CT values obtained with spit (23.51 [SD, 4.57]; P = 0.11) and sputum (25.82 [SD, 9.21]; P = 0.28) specimens were close to those of NPS. All alternative specimen collection methods were as sensitive as NPS, but spit collection appeared more promising, with a low CT value and ease of collection. Our findings warrant further investigation. IMPORTANCE Control of the COVID-19 pandemic relies heavily on a test-trace-isolate strategy. The most commonly used specimen for diagnosis of SARS-CoV-2 infection is a nasopharyngeal swab. However, this method is quite uncomfortable for the patient, requires specific equipment (nose swabs and containers), and requires close proximity to health care workers, putting them at risk of infection. Developing alternative sampling strategies could decrease the burden for health care workers, help overcome potential shortages of equipment, and improve acceptability of testing by reducing patient discomfort.

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Procedures for collection of induced sputum specimens from children.

Publications

Procedures for collection of induced sputum specimens from children.

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