Indian Council of Medical Research (ICMR), New Delhi, India.
Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh, India.
Mycoses. 2024 May;67(5):e13730. doi: 10.1111/myc.13730.
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.
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.
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.
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.
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.
由于传统技术诊断的延迟和高死亡率，开发一种标准化和快速的非培养技术是肺部、胃肠道和播散性毛霉菌病未满足的需求。尽管已经进行了一些有限的分子诊断研究，但对于这种高度致命的感染还没有建立血清学检测方法。
利用根毛霉的抗原开发和评估一种间接的酶联免疫吸附试验（ELISA），以检测毛霉菌病患者血清中的抗根毛霉抗体（IgG 和 IgM）。
我们使用标准化方案提取分泌型和菌丝型根毛霉抗原。Bradford 法用于蛋白质定量。然后，我们使用根毛霉菌丝和分泌型抗原（碳酸氢盐缓冲液 pH 9.2 中的 10.0 μg/mL）标准化间接 ELISA，以检测患者血清中的抗根毛霉 IgG 和 IgM 抗体。我们纳入了毛霉菌病、其他真菌感染和健康对照组的患者。为每个患者样本计算抗体指数值（E 值）。
使用 85%硫酸铵盐分级法从天门冬酰胺肉汤培养液滤液和酵母提取物蛋白胨葡萄糖（YPD）肉汤中培养的菌丝体用三氯乙酸（TCA）沉淀，得到了大量用于检测的高质量蛋白质。我们纳入了 55 名毛霉菌病患者（鼻眶脑毛霉菌病 [ROCM，n=39]、肺部 [n=15]、胃肠道 [n=1]）、24 名其他真菌感染患者（可能的曲霉病 [n=14]、念珠菌病 [n=10]）和 16 名健康对照组。该抗体试验诊断毛霉菌病的敏感性范围为 IgG 为 83.6-92.7%，IgM 为 72.7-87.3%，特异性为 IgG 为 91.7-92.5%，IgM 为 80-82.5%。其他真菌感染患者和健康个体的血清没有明显的交叉反应。
与基于 IgM 的 ELISA 相比，检测抗根毛霉 IgG 抗体在诊断 ROCM（敏感性为 84.6%比 69.2%）和肺部病例（敏感性为 86.6%比 80.0%）方面表现更好。需要进一步开展广泛的研究来证实我们的发现。
