Jaiswal Priyank, Datta Souvik, Sardar Bikash, Chaudhuri Surya Jyoti, Maji Dipankar, Ghosh Manab, Saha Bibhuti, Mukhopadhyay Sumi
Department of Laboratory Medicine, School of Tropical Medicine, West Bengal, India.
Department of Tropical Medicine, School of Tropical Medicine, Government of West Bengal, West Bengal, India.
PLoS One. 2018 Feb 8;13(2):e0192302. doi: 10.1371/journal.pone.0192302. eCollection 2018.
Post Kala Azar Dermal Leishmaniasis (PKDL) occurs as dermal consequence of previous Visceral Leishmaniasis (VL) infection and serves as an important reservoir for transmission of VL. Diagnosis of PKDL is often challenging for its symptomatic resemblance to other co-endemic diseases like Leprosy or Vitiligo. Parasitological examination by slit-skin smear and culture are the standard methods but lack high sensitivity. Thus, for efficient control of VL, reliable diagnostic and prognostic assay of PKDL are required.
Previously, glycoproteins (9-OAcSA) have been reported as promising biomarkers of Indian VL patients. However, till date, the status of glycans in Indian PKDL patients remains unexplored. Accordingly, in this study, the glyco-profile of PKDL Circulating Immune Complexes (CICs) as compared to other cross diseases like Vitiligo and Leprosyhas been investigated. Further, a novel Glyco CIC assay has been developed for efficient Indian PKDL patient diagnosis.
METHODS/PRINCIPAL FINDING: In the present study, 90 PKDL patients were enrolled from 3 VL endemic districts of West Bengal during 2015-16. Glycosylation profile of isolated CICs from sera of PKDL patients were initially analyzed through gradient SDS gel electrophoresis followed by PAS silver double staining, which revealed the presence of several glycan rich PKDL specific proteins of varying molecular weights. To further characterize the glyco-profile of acid dissociated affinity purified immuno-reactive antigens present in the CICs, glycosylation was demonstrated in these purified CIC antigens by DIG glycan differentiation kit with or without glycosidase as well as neuraminidase treatment. Diagnostic evaluation of the newly developed colorimetric Glyco CIC assay through Receiver Operating Characteristic (ROC) curve analysis revealed excellent (0.99) AUC value as compared to other conventional serodiagnostic assays like PEG CIC, Parasite ELISA (IgG and IgM). Additionally, longitudinal monitoring of 18 PKDL patients further revealed its good prognostic utility.
These results highlight the glycosylation status of CICs among Indian PKDL patients present in all the studied endemic districts of West Bengal. These PKDL biomarkers were completely absent in cross diseases like Vitiligo and Leprosy. Further, the newly developed Glyco CIC assay had an improved sensitivity of 95.6%, specificity of 99.3%, NPV of 97.1% and PPV of 98.9%.
黑热病后皮肤利什曼病（PKDL）是先前内脏利什曼病（VL）感染的皮肤后果，是VL传播的重要储存宿主。PKDL的诊断通常具有挑战性，因为其症状与麻风病或白癜风等其他共同流行疾病相似。通过皮肤涂片和培养进行寄生虫学检查是标准方法，但缺乏高灵敏度。因此，为了有效控制VL，需要可靠的PKDL诊断和预后检测方法。
此前，糖蛋白（9-OAcSA）已被报道为印度VL患者有前景的生物标志物。然而，迄今为止，印度PKDL患者中聚糖的状况仍未得到探索。因此，在本研究中，研究了与白癜风和麻风病等其他交叉疾病相比，PKDL循环免疫复合物（CIC）的糖谱。此外，还开发了一种新型糖基化CIC检测方法，用于高效诊断印度PKDL患者。
方法/主要发现：在本研究中，2015 - 2016年期间从西孟加拉邦的3个VL流行区招募了90名PKDL患者。最初通过梯度SDS凝胶电泳，然后进行PAS银双重染色，分析PKDL患者血清中分离出的CIC的糖基化谱，结果显示存在几种不同分子量的富含聚糖的PKDL特异性蛋白。为了进一步表征CIC中存在的酸解离亲和纯化免疫反应性抗原的糖谱，通过DIG聚糖分化试剂盒，在有或没有糖苷酶以及神经氨酸酶处理的情况下，对这些纯化的CIC抗原进行糖基化检测。通过受试者工作特征（ROC）曲线分析对新开发的比色糖基化CIC检测方法进行诊断评估，结果显示与其他传统血清学诊断方法如PEG CIC、寄生虫ELISA（IgG和IgM）相比，其曲线下面积（AUC）值优异（0.99）。此外，对18名PKDL患者的纵向监测进一步显示了其良好的预后效用。
这些结果突出了西孟加拉邦所有研究流行区中印度PKDL患者CIC的糖基化状态。这些PKDL生物标志物在白癜风和麻风病等交叉疾病中完全不存在。此外，新开发的糖基化CIC检测方法的敏感性提高到95.6%，特异性为99.3%，阴性预测值为97.1%，阳性预测值为98.9%。
