Department of Medical Microbiology, PGIMER, Chandigarh, India.
Advance Eye Center, PGIMER, Chandigarh, India.
Mycoses. 2023 Sep;66(9):801-809. doi: 10.1111/myc.13618. Epub 2023 Jun 25.
To compare the performance of conventional, semi-nested and real-time panfungal ITS PCRs for diagnosing fungal keratitis (FK) and develop genus-specific real-time PCR for the most common aetiology of FK.
This multicentric study includes 232 corneal samples from suspected FK patients from four centres across India between November 2019 through August 2021. A total of 87 corneal buttons were included for the comparison of conventional, semi-nested and real-time ITS PCRs, of which 68 were from confirmed FK patients. Of these 87 samples, 44 (microscopy and culture positive for Aspergillus sp. and/or Fusarium sp.) were used for the standardisation of genus-specific real-time primers/probes. Subsequently, the best method showing highest sensitivity and specificity was validated in 188 samples.
On Bayesian comparison, conventional ITS2 PCR showed best performance (sensitivity and specificity of 55.88% and 100%, respectively). Since, real-time ITS2 PCR was also considerably efficient (sensitivity and specificity of 51.47% and 84.21%, respectively) in comparison with the conventional PCR but faster, cost-effective, and less labor-intensive, ITS-2 real-time PCR is a suitable method that can be applied along with culture and microscopy. During validation, real-time PCR with genus-specific primers showed 61.76% and 91.18% sensitivity with specificity of 98.05% and 79.22%, respectively, for Aspergillus sp. and Fusarium sp. Aspergillus probe, Fusarium probe and duplex PCR showed sensitivity of 52.94%, 50% and 54.41% with specificity of 92.86%, 82.47% and 75%, respectively. No cross-reactivity of genus-specific PCRs was observed during standardisation.
ITS-2 real-time PCR can be applied as an adjunct with conventional methods for the diagnosis of FK. The genus-specific duplex real-time PCRs are rapid which reduces the turnaround time (TAT) avoiding the need for sequencing.
为了比较常规、半巢式和实时泛真菌 ITS PCR 在诊断真菌性角膜炎（FK）方面的性能，并为 FK 最常见的病因开发种特异性实时 PCR。
本多中心研究包括 2019 年 11 月至 2021 年 8 月印度四个中心的 232 例疑似 FK 患者的角膜样本。共有 87 个角膜纽扣用于比较常规、半巢式和实时 ITS PCR，其中 68 个来自确诊 FK 患者。在这 87 个样本中，44 个（显微镜和培养阳性为曲霉属和/或镰刀菌属）用于种特异性实时引物/探针的标准化。随后，在 188 个样本中验证了显示最高灵敏度和特异性的最佳方法。
贝叶斯比较显示，常规 ITS2 PCR 表现最佳（灵敏度和特异性分别为 55.88%和 100%）。由于实时 ITS2 PCR 与常规 PCR 相比也相当有效（灵敏度和特异性分别为 51.47%和 84.21%），但速度更快、更具成本效益且劳动强度更低，ITS-2 实时 PCR 是一种合适的方法，可与培养和显微镜一起应用。在验证过程中，种特异性引物的实时 PCR 对曲霉属和镰刀菌属的灵敏度分别为 61.76%和 91.18%，特异性分别为 98.05%和 79.22%。在标准化过程中，种特异性 PCR 未观察到交叉反应。
ITS-2 实时 PCR 可作为常规方法的辅助手段用于 FK 的诊断。种特异性实时双重 PCR 快速，可缩短周转时间（TAT），避免测序的需要。
