Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.
Optom Vis Sci. 2021 Mar 1;98(3):280-284. doi: 10.1097/OPX.0000000000001652.
The significance of the study is that, although conventional culture remains the criterion standard for identifying the causative fungal pathogens, polymerase chain reaction (PCR) may serve as a powerful and high-throughput tool for the early and definitive diagnosis of high-risk patients with mycotic keratitis owing to high sensitivity and specificity.
This study was focused on comparing the results of PCR with traditional microbial studies for the detection and identification of fungal pathogens in patients with clinically suspected fungal keratitis.
Corneal scrapings were collected from 59 patients with clinically suspected fungal keratitis for routine culture, staining, and seminested PCR assay for fungal pathogen identification. The results of PCR were compared with a conventional microbial workup (smear and culture). The samples that were unidentified by culture but were amplified by PCR were further identified by nucleotide sequencing.
Of the 59 patients with suspected fungal keratitis, 38 (64.40%) were found to be positive by PCR assay, 24 (40.67%) by culture, 18 (20.3%) by potassium hydroxide wet mount, and 8 (13.5%) by Gram stains for fungal pathogens. All the 24 isolates found positive with culture were also positive with PCR, so they had not been sequenced for molecular identification. The remaining 14 isolates that were positive with PCR but negative with culture were further identified as Cladosporium cladosporioides, Simplicillium species, Fusarium solani, Alternaria tenuissima, Chaetomium globosum, Penicillium citrinum, and Rhizopus microsporus by sequencing up to the species level.
The PCR was able to detect the presence of fungal pathogens in a high proportion of culture-negative cases. This study suggests that PCR may serve as a rapid, important complement to traditional culture with high-throughput means of fungal pathogen identification in patients with clinically suspected fungal keratitis.
该研究的意义在于，尽管传统培养仍然是鉴定致病真菌病原体的标准，但聚合酶链反应（PCR）可能成为一种强大且高通量的工具，用于对高危真菌角膜炎患者进行早期和明确诊断，因为它具有高灵敏度和特异性。
本研究旨在比较 PCR 与传统微生物研究在检测和鉴定临床上疑似真菌性角膜炎患者中的真菌病原体方面的结果。
从 59 例临床上疑似真菌性角膜炎患者的角膜刮片中采集标本，进行常规培养、染色和半巢式 PCR 检测以鉴定真菌病原体。将 PCR 结果与常规微生物检查（涂片和培养）进行比较。通过培养无法鉴定但通过 PCR 扩增的样本，通过核苷酸测序进一步鉴定。
在 59 例疑似真菌性角膜炎患者中，38 例（64.40%）通过 PCR 检测呈阳性，24 例（40.67%）通过培养，18 例（20.3%）通过氢氧化钾湿片，8 例（13.5%）通过革兰氏染色检测真菌病原体呈阳性。所有通过培养发现的 24 个阳性分离株也与 PCR 呈阳性，因此未对其进行分子鉴定测序。另外 14 个通过 PCR 检测呈阳性但培养呈阴性的分离株通过测序进一步鉴定为枝孢属、丛梗孢属、茄病镰刀菌、细极链格孢菌、球毛壳菌、桔青霉和微小根毛霉。
PCR 能够检测出培养阴性病例中存在真菌病原体。本研究表明，PCR 可能成为一种快速、重要的补充手段，与传统培养相结合，通过高通量手段鉴定临床上疑似真菌性角膜炎患者的真菌病原体。
