PCR and culture for diagnosis ofAcanthamoebakeratitis

Abstract

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

Acanthamoeba Keratitis, Pathology, Diagnosis and Treatment

“…Performing a corneal scraping for microbial culture to identify the causative pathogen(s) is generally considered as the gold standard clinically to confirm AK. Although culture tests carry a high specificity (100%), they generally lack strength regarding sensitivity, which ranges from 7–66.7% depending upon culture techniques using agar culturing plates with or without overlay of feeding bacteria [ 36 , 37 ]. This makes the test statistically weak for determining AK as a diagnosis.…”

“…Another aspect to consider is the time that is required to get results back from a culture. Amoebic culture takes, on average, 10 days to demonstrate a positive result [ 36 ]. This delay allows clinical morbidities to further progress, making AK more difficult to treat.…”

“…PCR is not used clinically as broadly as corneal culture; however, the diagnostic tool does improve upon statistical strength for diagnosis of AK. PCR demonstrates a high specificity (100%), as well as a moderately better sensitivity than corneal culture, which ranges from 66.7–100% depending on the DNA section used [ 36 , 37 ]. These statistical values trend toward the higher end if multiple PCR tests are run using different DNA segments.…”

“…These statistical values trend toward the higher end if multiple PCR tests are run using different DNA segments. PCR also provides faster results, with the average turnover in diagnosis being 5 days [ 36 ]. Performing PCR is a moderately technical process and can be more expensive than other diagnostic techniques.…”

“…Real time PCR is a newer technique that allows for even faster results. While real time PCR carries a similar range of sensitivity and specificity as standard PCR, as well as a high negative predictive value (99.3%), positive predictive values of real time PCR are relatively low (59.1–61.9%) [ 36 ]. The technique also requires specialized machinery.…”