Background

Acyclovir (ACV) is the most commonly used drug for herpes simplex virus (HSV) infection therapy. Prolonged antiviral therapy or prophylaxis in immunocompromised patients may promote the development of drug-resistant strains. Due to the high polymorphism in genes involved in drug resistance, phenotypic methods, although work-intensive, are still required to test drug susceptibility. Real-time cell analysis (RTCA) based methods could offer a rapid and less labor-intensive alternative for phenotypic testing of ACV resistance.

Objective

To investigate the utility of a new RTCA based assay (RTCAA) to test acyclovir susceptibility of HSV clinical isolates.

Study design

Four reference strains and 93 clinical isolates (60 HSV-1 and 33 HSV-2) were tested by RTCAA. In the presence of ACV concentrations from 2.2 to 140.8 µM, Vero cells were infected with different virus dilutions. IC₅₀ values were calculated by dose-response curve (DRC) with area-under-curve (AUC) method. The reference strains and 22 clinical isolates were additionally tested by dye-uptake assay, and IC₅₀ values of both methods were compared.

Results

IC₅₀ values from RTCAA and dye-uptake assays were positively correlated (Spearman's rho = 0.897, p < 0.001) and quantitatively agreed (Bland-Altman plot). Based on a cut-off of 4 µM for HSV-1 and 13 µM for HSV-2, 87 isolates were classified as ACV-sensitive and 6 isolates as ACV-resistant. The reference strains showed the expected results of ACV susceptibility.

Conclusion

RTCAA agrees well with the dye-uptake assay. Compared with other phenotypic methods, RTCAA requires less manipulation, reduces the workload and the turnaround time, and appears to be an objective and reliable method to test ACV susceptibility.

中文翻译：

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通过基于实时细胞分析的新方法对患者1型和2型单纯疱疹病毒分离株的阿昔洛韦耐药性进行表型测试。

背景

阿昔洛韦（ACV）是用于单纯疱疹病毒（HSV）感染治疗的最常用药物。对免疫功能低下的患者进行长期抗病毒治疗或预防可能会促进耐药菌株的产生。由于涉及耐药性的基因具有很高的多态性，因此尽管需要大量工作，但表型方法仍需要测试药物敏感性。基于实时细胞分析（RTCA）的方法可以为ACV抗性的表型测试提供快速且省力的替代方法。

目的

调查新的基于RTCA的检测方法（RTCAA）的效用，以测试HSV临床分离株对阿昔洛韦的敏感性。

学习规划

RTCAA检测了4株参考菌株和93株临床分离株（60株HSV-1和33株HSV-2）。在ACV浓度为2.2至140.8 µM的情况下，用不同的病毒稀释液感染Vero细胞。通过剂量反应曲线（DRC）和曲线下面积（AUC）方法计算IC ₅₀值。参考菌株和22种临床分离株还通过染料吸收试验进行了测试，并比较了两种方法的IC ₅₀值。

结果

RTCAA和染料吸收测定的IC ₅₀值呈正相关（Spearman的rho = 0.897，p <0.001），并且定量一致（Bland-Altman图）。根据HSV-1的截止值为4 µM，HSV-2的截止值为13 µM，将87个分离株归为对ACV敏感，将6个分离株归为对ACV耐药。参考菌株显示了ACV敏感性的预期结果。

结论

RTCAA与染料吸收测定非常吻合。与其他表型方法相比，RTCAA所需的操作更少，减少了工作量并缩短了周转时间，并且似乎是测试ACV敏感性的客观可靠的方法。