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Published on: December 22, 2014
Impact of Sample Collection Order on the Diagnostic Performance of Metagenomic Deep Sequencing for Infectious
Travis K Redd1,2, Prajna Lalitha3, N Venkatesh Prajna3
1Francis I. Proctor Foundation, University of California San Francisco, CA.
Purpose:
The purpose of this article was to evaluate the impact of sample collection order on the diagnostic yield of metagenomic deep sequencing (MDS) for determining the causative pathogen in infectious keratitis.
Methods:
We performed a cross-sectional diagnostic test evaluation among subjects with infectious keratitis at Aravind Eye Hospital in Madurai, India. All subjects underwent corneal scrapings of the affected eye to obtain potassium hydroxide smear, Gram stain, bacterial culture, and fungal culture, in this order. The order of MDS specimen collection relative to smear and culture samples was randomized and served as the primary predictor. Outcomes included the normalized copy number of pathogenic RNA detected by MDS, the proportion of MDS samples that were diagnostic, and the agreement of MDS results with cultures.
Results:
MDS samples from 46 subjects with corneal ulcers were evaluated. MDS was positive in 33 subjects (76%) and had 74% overall agreement with culture results. There was no association between order of MDS sample collection and normalized copy number of genetic material detected (P = 0.62) or the likelihood of MDS positivity (P = 0.46). However, the likelihood of agreement between MDS and cultures decreased when MDS corneal swabs were collected after other diagnostic corneal scrapings (P = 0.05).
Conclusions:
The overall yield of MDS for detecting the cause of infectious keratitis was not affected by sample collection order. However, diagnostic agreement between MDS and cultures decreased when MDS samples were collected after other specimens. Additional investigation is warranted to determine whether this represents increased sensitivity of MDS compared with cultures or higher susceptibility to contaminants.
Insights
Sample collection order did not impact metagenomic deep sequencing (MDS) yield for infectious keratitis. However, collecting MDS samples after other tests reduced agreement with culture results, warranting further investigation.
Area of Science:
- Ophthalmology
- Microbiology
- Genetics
Background:
- Infectious keratitis diagnosis relies on identifying causative pathogens.
- Metagenomic deep sequencing (MDS) offers a culture-independent diagnostic approach.
- The impact of sample collection methodology on MDS diagnostic yield is not fully understood.
Purpose of the Study:
- To evaluate how the order of sample collection affects the diagnostic yield of metagenomic deep sequencing (MDS).
- To determine the impact of sample collection order on pathogen identification in infectious keratitis.
Main Methods:
- A cross-sectional diagnostic test evaluation was conducted.
- Subjects with infectious keratitis underwent corneal scrapings for smear, Gram stain, bacterial, and fungal cultures.
- The order of MDS specimen collection relative to other samples was randomized.
Main Results:
- Metagenomic deep sequencing (MDS) was positive in 76% of subjects.
- There was no association between MDS sample collection order and the amount of genetic material detected.
- Agreement between MDS and culture results decreased when MDS samples were collected after other diagnostic specimens (P=0.05).
Conclusions:
- The overall diagnostic yield of MDS for infectious keratitis was not influenced by sample collection order.
- A decreased agreement between MDS and cultures was observed when MDS samples were collected last.
- Further research is needed to ascertain if this reflects MDS sensitivity or contamination.
