Detection and Identification ofAcanthamoebaand Other Nonviral Causes of Infectious Keratitis in Corneal Scrapings by Real-Time PCR and Next-Generation Sequencing-Based 16S-18S Gene Analysis

Abstract

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 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 using specific real-time PCR, 21 of which were positive using 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). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype 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 non-viral causes of IK, including Acanthamoeba.

Entamoeba gingivalis: epidemiology, genetic diversity and association with oral microbiota signatures in North Eastern Tanzania

“…DNA was quantified using the Quant-IT high-sensitivity double-stranded DNA (dsDNA) assay kit (Thermo Fisher Scientific), and the PCR products were pooled in equimolar amounts from the individual samples into the pooled amplicon library (PAL). Undesirable DNA amplicons were removed from the PAL using Agencourt AMPure XP Bead (Beckman Coulter)-based purification as previously described [ 16 ]. The resulting AMPure bead-purified PAL was diluted to its final concentration of 11.5 pM DNA with a 0.001 N NaOH concentration and used for sequencing on the Illumina MiSeq desktop sequencer (Illumina Inc., San Diego, CA, USA).…”

“…Genomic DNAs extracted from the mouthwash samples were subject to microbiota profiling (metabarcoding), using the recently described 16S + 18S assay in place at Statens Serum Institut [16][17][18]. Briefly, PCR used one primer pair targeting the 16S (341F3/ 806R5 [5ʹ-ACT CCT AYG GGR BGC ASC AG-3ʹ/5ʹ-AGC GTG GAC TAC NNG GGT ATC TAA T-3ʹ]) and three primer pairs targeting the 18S (G3F1/ G3R1 [5ʹ-GCC AGC AGC CGC GGT AAT TC-3ʹ/5ʹ-ACA TTC TTG GCA AAT GCT TTC GCA G-3ʹ], G4F3/G4R3 [5ʹ-CAG CCG CGG TAA TTC CAG CTC-3ʹ/5ʹ-GGT GGT GCC CTT CCG TCA AT-3ʹ], and G6F1/G6R1 [3ʹ-TGG AGG GCA AGT CTG GTG CC-3ʹ/5ʹ-ACG GTA TCT GAT CGT CTT CGA TCC C-3ʹ]).…”

Parasitic Intestinal Protists of Zoonotic Relevance Detected in Pigs by Metabarcoding and Real-Time PCR

“…The amplicon-based NGS-based approach to detecting and differentiating parasites used in this study has been applied in a number of studies involving human, animal, and environmental samples, including corneal scrapings, fecal samples, and sewage samples [14,15,22,33], and with different foci from clinically relevant opportunistic parasites to foodborne parasites [34]. This 'one-fits-many' approach could be cost-effective for screening DNAs from large numbers of samples for DNA from parasites, fungi, and bacteria, although some limitations have been identified [15,34].…”

“…The DNAs were processed by the metabarcoding assay [12][13][14][15]22]. This method involves PCR-based amplification (PCR 1) of ribosomal genes using one set of primers targeting 16S and three sets of primers targeting 18S (Table 1).…”

“…As an example, we previously introduced a metabarcoding assay relying on amplicon-based NGS of nuclear ribosomal genes from bacteria, fungi, and parasites with automated software-based annotation of sequences to genus and-oftentimesspecies/sub-species level. This method has proven useful for screening various matrices such as skin samples, cornea scrapings, sewage samples, and human stool samples for parasites and other non-viral organisms, the relevance of which depends on the focus and scope of the investigation [12][13][14][15].…”