Shimoyama Ken, Kan Shigenori, Takahashi Gaku, Morino Gota, Yamada Yasuhiko, Inoue Yoshihiro, Inada Katsuya, Endo Shigeatsu
Department of Critical Care Medicine, School of Medicine, Iwate Medical University, Iwate Prefecture, Japan.
Morioka Yuai Hospital, Iwate Prefecture, Japan.
Infect Chemother. 2021 Mar;53(1):75-83. doi: 10.3947/ic.2020.0128.
Currently, supplementary serological testing for β-D glucan (BDG) is often selected to diagnose deep mycosis in care covered by the health insurance in Japan. The Wako method used by our center has low sensitivity, and different studies have used different cut-off values due to factors that cause false positives and false negatives. One possible cause of false negatives is the use of platelet-rich plasma (PRP) as the sample material. Because phagocytic white blood cells (WBC) are precipitated by centrifugation and only plasma is measured, it seems unlikely that the actual amount of BDG is being measured when using PRP. Further, a frequent cause of false positives is contamination from blood products and gauze containing BDG. To resolve these issues, the blood cell separator, hydroxyethyl starch, is used to precipitate only the red blood cells to obtain leukocyte-rich plasma (LRP). We hypothesized that it might be possible to improve the diagnostic rate of deep mycosis by measuring the BDG content of plasma containing WBC and fungal components and by comparing the BDG content of PRP and LRP measured simultaneously.
Healthy human blood, albumin-added blood, wrung-out gauze fluid-added blood, and fungal solution-added blood were prepared, and PRP and LRP were prepared using hydroxyethyl starch. The BDG content of each sample was measured using the Wako method and compared. In addition, PRP and LRP of fungal-added blood were Gram-stained and examined under a microscope, and the number of WBCs and phagocytosed fungi was counted visually and compared.
Measuring the BDG content of LRP confirmed that there were no false positives with LRP, and experiments comparing albumin-added false-positive blood to fungal-added blood showed significant differences between PRP and LRP only in the fungal-added blood.
Calculating the BDG-ratio (LRP/PRP) by measuring both LRP and PRP may eliminate false positives and false negatives of true deep mycosis and improve the diagnostic rate.
目前，在日本医疗保险覆盖的医疗中，β-D葡聚糖（BDG）的补充血清学检测常被用于诊断深部真菌病。我们中心使用的和光方法灵敏度较低，并且由于导致假阳性和假阴性的因素，不同研究采用了不同的临界值。假阴性的一个可能原因是使用富含血小板血浆（PRP）作为样本材料。因为吞噬性白细胞（WBC）通过离心沉淀，仅测量血浆，所以使用PRP时似乎不太可能测量到BDG的实际含量。此外，假阳性的常见原因是来自含BDG的血液制品和纱布的污染。为解决这些问题，使用血细胞分离器羟乙基淀粉仅沉淀红细胞以获得富含白细胞血浆（LRP）。我们假设通过测量含有白细胞和真菌成分的血浆中的BDG含量，并比较同时测量的PRP和LRP的BDG含量，可能提高深部真菌病的诊断率。
制备健康人血液、添加白蛋白的血液、添加拧干纱布液的血液和添加真菌溶液的血液，并使用羟乙基淀粉制备PRP和LRP。使用和光方法测量每个样本的BDG含量并进行比较。此外，对添加真菌的血液的PRP和LRP进行革兰氏染色并在显微镜下检查，目视计数白细胞和吞噬真菌的数量并进行比较。
测量LRP的BDG含量证实LRP无假阳性，将添加白蛋白的假阳性血液与添加真菌的血液进行比较的实验表明，PRP和LRP之间仅在添加真菌的血液中有显著差异。
通过同时测量LRP和PRP来计算BDG比值（LRP/PRP）可能消除真正深部真菌病的假阳性和假阴性并提高诊断率。
