Cassels Kathryn, Almofeez Raghad, Roman Jessica, Steinberg Hannah, Byne Ahana, Haymond Amanda, Tinajeros Freddy, Del Carmen Menduiña María, Málaga Machaca Edith, Verástegui Manuela, Ramírez José Luis, Liotta Lance, Gilman Robert H, Luchini Alessandra
Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, United States of America.
Department of Microbiology and Immunology, University of Illinois Chicago, Chicago, Illinois, United States of America.
PLoS Negl Trop Dis. 2025 Jun 16;19(6):e0013082. doi: 10.1371/journal.pntd.0013082. eCollection 2025 Jun.
Caused by the parasite Trypanosoma cruzi, Chagas disease affects an estimated 7 million people globally. Diagnosis of Chagas disease in infants is urgently needed, as early detection allows for more effective treatment and reduced mortality. However, current diagnostics are inappropriate for effective detection in infants due to differences in the mechanism of disease in infants and the infant immune system, as well as lack of diagnostic sensitivity and loss to follow up. Studying peripheral biomarkers in urine can leverage physiological concentration in the bladder to increase yield of proteins secreted by pathogen, infected cells, or antigen processed by immune cells residing in different body sites.
We analyzed the urine of a cohort of infants who were congenitally infected with Chagas disease, using a method including affinity enrichment, mass spectrometry, and bioinformatics analysis to characterize the T. cruzi secreted peptidome. We identified 198 peptides specific for T. cruzi and analyzed them in light of their potential for diagnostic utility. Our protocol revealed that peptides of the hyper-mutating mucin-associated surface protein and trans-sialidase protein families could be identified in patient urine and can serve as diagnostic markers of disease. We developed antibodies against conserved regions of each protein and validated that these antibodies could be used to differentiate the urine of Chagas disease patients (N = 16 cases) from healthy controls (N = 19). By utilizing affinity enrichment sample preprocessing and anti-trans-sialidase and anti-MASP antibodies in tandem, we differentiated cases from controls with 87.5% sensitivity and 94.7% specificity.
CONCLUSIONS/SIGNIFICANCE: Our work suggests that it is possible to detect Trypanosoma cruzi infection directly from a noninvasively collected fluid such as urine. A direct test in urine with this success rate would be well suited for rapid diagnosis in low-resource areas. Further studies to validate this approach are warranted.
恰加斯病由克氏锥虫寄生虫引起，全球约有700万人受其影响。迫切需要对婴儿进行恰加斯病诊断，因为早期检测可实现更有效的治疗并降低死亡率。然而，由于婴儿疾病机制和免疫系统的差异，以及缺乏诊断敏感性和随访失访情况，目前的诊断方法不适用于婴儿的有效检测。研究尿液中的外周生物标志物可以利用膀胱中的生理浓度，提高病原体、感染细胞或驻留在不同身体部位的免疫细胞处理的抗原所分泌蛋白质的产量。
我们分析了一组先天性感染恰加斯病的婴儿的尿液，使用包括亲和富集、质谱和生物信息学分析在内的方法来表征克氏锥虫分泌的肽组。我们鉴定出198种克氏锥虫特异性肽，并根据其诊断效用潜力对其进行了分析。我们的方案显示，高突变粘蛋白相关表面蛋白和转唾液酸酶蛋白家族的肽可以在患者尿液中鉴定出来，并可作为疾病的诊断标志物。我们开发了针对每种蛋白质保守区域的抗体，并验证这些抗体可用于区分恰加斯病患者（N = 16例）和健康对照（N = 19例）的尿液。通过串联使用亲和富集样品预处理以及抗转唾液酸酶和抗MASP抗体，我们以87.5%的灵敏度和94.7%的特异性区分了病例和对照。
结论/意义：我们的工作表明，有可能直接从尿液等非侵入性采集的液体中检测克氏锥虫感染。这种成功率的尿液直接检测方法非常适合资源匮乏地区的快速诊断。有必要进行进一步的研究来验证这种方法。
