Dataset Information

Comparison of respiratory pathogen yields from Nasopharyngeal/Oropharyngeal swabs and sputum specimens collected from hospitalized adults in rural Western Kenya.

ABSTRACT: Molecular diagnostic methods are becoming increasingly available for assessment of acute lower respiratory illnesses (ALRI). However, nasopharyngeal/oropharyngeal (NP/OP) swabs may not accurately reflect etiologic agents from the lower respiratory tract where sputum specimens are considered as a more representative sample. The pathogen yields from NP/OP against sputum specimens have not been extensively explored, especially in tropical countries. We compared pathogen yields from NP/OP swabs and sputum specimens from patients ?18 years hospitalized with ALRI in rural Western Kenya. Specimens were tested for 30 pathogens using TaqMan Array Cards (TAC) and results compared using McNemar's test. The agreement for pathogen detection between NP/OP and sputum specimens ranged between 85-100%. More viruses were detected from NP/OP specimens whereas Klebsiella pneumoniae and Mycobacterium tuberculosis were more common in sputum specimens. There was no clear advantage in using sputum over NP/OP specimens to detect pathogens of ALRI in adults using TAC in the context of this tropical setting.

SUBMITTER: Nyawanda BO

PROVIDER: S-EPMC6677726 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Comparison of respiratory pathogen yields from Nasopharyngeal/Oropharyngeal swabs and sputum specimens collected from hospitalized adults in rural Western Kenya.

Nyawanda Bryan O BO Njuguna Henry N HN Onyango Clayton O CO Makokha Caroline C Lidechi Shirley S Fields Barry B Winchell Jonas M JM Katieno Jim S JS Nyaundi Jeremiah J Ade Fredrick F Emukule Gideon O GO Mott Joshua A JA Otieno Nancy N Widdowson Marc-Alain MA Chaves Sandra S SS

Scientific reports 20190802 1

Molecular diagnostic methods are becoming increasingly available for assessment of acute lower respiratory illnesses (ALRI). However, nasopharyngeal/oropharyngeal (NP/OP) swabs may not accurately reflect etiologic agents from the lower respiratory tract where sputum specimens are considered as a more representative sample. The pathogen yields from NP/OP against sputum specimens have not been extensively explored, especially in tropical countries. We compared pathogen yields from NP/OP swabs and ...[more]

PMID: 31375774

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Project description:BACKGROUND:Careful 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. RESULTS:We 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/?l) 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. CONCLUSIONS:We 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.

Dataset Information

Comparison of respiratory pathogen yields from Nasopharyngeal/Oropharyngeal swabs and sputum specimens collected from hospitalized adults in rural Western Kenya.

Publications

Comparison of respiratory pathogen yields from Nasopharyngeal/Oropharyngeal swabs and sputum specimens collected from hospitalized adults in rural Western Kenya.

Similar Datasets

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