TY - JOUR
T1 - Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis
AU - Holmgaard, Dennis Back
AU - Barnadas, Celine
AU - Mirbarati, Seyed Hossein
AU - Andersen, Lee O'Brien
AU - Nielsen, Henrik Vedel
AU - Stensvold, Christen Rune
N1 - Copyright © 2021 Holmgaard et al.
PY - 2021/2
Y1 - 2021/2
N2 -
Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of
Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for the detection of
Acanthamoeba Two hundred DNAs extracted from corneal scrapings and screened by
Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive by specific real-time PCR, of which 21 were positive by the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (
n = 19) and T6 (
n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for
Acanthamoeba, exemplified by
Pseudomonas aeruginosa (
n = 11),
Moraxella spp. (
n = 6),
Staphylococcus aureus (
n = 2),
Fusarium spp. (
n = 4), and
Candida albicans (
n = 1). In conclusion, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting
Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotypes was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for
Acanthamoeba NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting nonviral causes of IK, including
Acanthamoeba.
AB -
Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of
Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for the detection of
Acanthamoeba Two hundred DNAs extracted from corneal scrapings and screened by
Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive by specific real-time PCR, of which 21 were positive by the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (
n = 19) and T6 (
n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for
Acanthamoeba, exemplified by
Pseudomonas aeruginosa (
n = 11),
Moraxella spp. (
n = 6),
Staphylococcus aureus (
n = 2),
Fusarium spp. (
n = 4), and
Candida albicans (
n = 1). In conclusion, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting
Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotypes was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for
Acanthamoeba NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting nonviral causes of IK, including
Acanthamoeba.
KW - Denmark
KW - keratitis
KW - NGS
KW - ocular disease
KW - microbiome
KW - parasite infection
U2 - 10.1128/JCM.02224-20
DO - 10.1128/JCM.02224-20
M3 - Article
C2 - 33239372
VL - 59
JO - Journal of Clinical Microbiology
JF - Journal of Clinical Microbiology
SN - 0095-1137
IS - 2
ER -