Abstract：
BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection.
OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis.
METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 μg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample.
RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity.
CONCLUSIONS: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.
OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis.
METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 μg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample.
RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity.
CONCLUSIONS: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.
摘要：
背景：由于常规技术的诊断延迟和高死亡率，标准化和快速的非基于培养的技术的发展是肺部未满足的需求，胃肠,和播散形式的毛霉菌病。尽管对分子诊断进行了有限的研究，对于这种高度致命的感染，没有确定的血清学测试。
目的：建立并评估一种间接的内部酶联免疫吸附试验（ELISA），该试验利用阿根根霉抗原检测患者血清中抗根霉抗体（IgG和IgM）。
方法：我们使用标准化方案提取了分泌型和菌丝体根霉抗原。Bradford测定法用于蛋白质定量。然后，我们使用R.arhizus菌丝体和分泌抗原（在碳酸氢盐缓冲液pH9.2中的10.0μg/mL）对间接ELISA进行了标准化，以检测患者血清中的抗根霉IgG和IgM抗体。我们纳入了毛霉菌病患者，其他真菌感染，和健康的控制。计算每个患者样品的抗体指数值(E值)。
结果：天冬酰胺肉汤培养滤液利用85％硫酸铵盐分级分离和在用三氯乙酸（TCA）沉淀的酵母提取物蛋白胨葡萄糖（YPD）肉汤中生长的菌丝体匀浆产生了大量优质的蛋白质用于测定。我们纳入了55例毛霉菌病患者（犀牛-大脑毛霉菌病[ROCM，n=39]，肺[n=15]，胃肠[n=1])，24患有其他真菌感染（可能的曲霉病[n=14]，念珠菌病[n=10])，和健康对照（n=16）。抗体检测诊断毛霉菌病的敏感性,IgG为83.6-92.7%,IgM为72.7-87.3%,IgG的特异性为91.7-92.5%，IgM的特异性为80-82.5%。来自患有其他真菌感染的患者和健康个体的血清没有显示出显著的交叉反应性。
结论：与基于IgM的ELISA相比，抗根霉IgG抗体的检测在诊断两种ROCM方面均明显更好（敏感性为84.6％vs.69.2％）和肺部病例（86.6％vs.80.0%）。需要更广泛的研究来证实我们的发现。
目的：建立并评估一种间接的内部酶联免疫吸附试验（ELISA），该试验利用阿根根霉抗原检测患者血清中抗根霉抗体（IgG和IgM）。
方法：我们使用标准化方案提取了分泌型和菌丝体根霉抗原。Bradford测定法用于蛋白质定量。然后，我们使用R.arhizus菌丝体和分泌抗原（在碳酸氢盐缓冲液pH9.2中的10.0μg/mL）对间接ELISA进行了标准化，以检测患者血清中的抗根霉IgG和IgM抗体。我们纳入了毛霉菌病患者，其他真菌感染，和健康的控制。计算每个患者样品的抗体指数值(E值)。
结果：天冬酰胺肉汤培养滤液利用85％硫酸铵盐分级分离和在用三氯乙酸（TCA）沉淀的酵母提取物蛋白胨葡萄糖（YPD）肉汤中生长的菌丝体匀浆产生了大量优质的蛋白质用于测定。我们纳入了55例毛霉菌病患者（犀牛-大脑毛霉菌病[ROCM，n=39]，肺[n=15]，胃肠[n=1])，24患有其他真菌感染（可能的曲霉病[n=14]，念珠菌病[n=10])，和健康对照（n=16）。抗体检测诊断毛霉菌病的敏感性,IgG为83.6-92.7%,IgM为72.7-87.3%,IgG的特异性为91.7-92.5%，IgM的特异性为80-82.5%。来自患有其他真菌感染的患者和健康个体的血清没有显示出显著的交叉反应性。
结论：与基于IgM的ELISA相比，抗根霉IgG抗体的检测在诊断两种ROCM方面均明显更好（敏感性为84.6％vs.69.2％）和肺部病例（86.6％vs.80.0%）。需要更广泛的研究来证实我们的发现。
