[Application of serum N -glycan profiling diagnostic model in evaluation of liver fibrosis in patients with hepatitis C].

Objective: To study the changes of serum N-glycan abundance in patients with liver fibrosis at different stages of hepatitis C, and to establish and evaluate the diagnostic model for clinical application value. Methods: Data of 169 hepatitis C virus-infected cases with liver fibrosis were enrolled. Nine kinds of serum N-glycans were detected and analyzed using DNA sequencer-assisted fluorophore-assisted capillary electrophoresis technology. A binary logistics regression method was used to establish a diagnostic model based on the changes in the relative content of N-glycans in each stage of liver fibrosis. Receiver operating characteristic curve was used to evaluate and compare the diagnostic efficacy with other liver fibrosis diagnostic models. Results: N-glycan diagnostic model (B and C) had highest AUROC= 0.776, 0.827 for distinguishing fibrosis S1~S2 to S3~S4 and S1~S3 to S4 than GlycoFibroTest (AUROC = 0.760, 0.807), GlycoCirrhoTest (AUROC = 0.722, 0.787), aspartate aminotransferase to platelet ratio index (AUROC = 0.755, 0.751), FIB-4 index (AUROC = 0.730, 0.774), and S-index (AUROC = 0.707, 0.744). However, the diagnostic efficacy of model A (AUROC = 0.752) for distinguishing fibrosis S1 with S2~S4 had lower diagnostic potency than that of the aspartate aminotransferase to platelet ratio index (AUROC = 0.807). Diagnostic efficiency was improved when the N-glycan profiling and the aspartate aminotransferase to platelet ratio index were combined to diagnose liver fibrosis in each stage, and the area under the receiver operating characteristic curve was 0.839, 0.825, and 0.837, respectively. Conclusion: The serum N-glycan profiling diagnostic model has potential clinical application value in the diagnosis of liver fibrosis in patients with hepatitis C.

Efficacies of two in-house indirect ELISAs for diagnosis of amoebic liver abscess.

Entamoeba histolytica causes amoebic diarrhoea, colitis and liver abscess (ALA). Diagnosis of ALA is difficult, as most patients do not have simultaneous intestinal amoebic infection. At Hospital Universiti Sains Malaysia (HUSM), diagnosis of ALA relies on a combination of clinical findings, ultrasound examination of the liver and serodiagnosis using a commercial kit. In this study, two in-house indirect ELISAs were developed and evaluated. One of the in-house assays utilises E. histolytica crude soluble antigen (CSA) to detect serum IgG specific to the parasite whereas the other uses E. histolytica ether extract antigen (EEA). Preparation of CSA requires a sonicator to lyse the amoeba whereas EEA was prepared by chemically solubilizing the trophozoites. Based on the cut-off value of mean optical density + 3SD, CSA-ELISA showed 100% (24/24) sensitivity and 93.33% (210/225) specificity; while EEA-ELISA showed 91.67% (22/24) sensitivity and 95.11% (214/225) specificity. In conclusion, both the in-house indirect ELISAs were found to be efficacious for diagnosis of ALA; and the EEA is easier to prepare than the commonly used CSA.

Identification of Entamoeba histolytica trophozoites in fresh stool sample: comparison of three staining techniques and study on the viability period of the trophozoites.

Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.

Seroprevalence of anti-amoebic antibody among blood donors by indirect hemeagglutination assay.

The screening for anti-amoebic antibody among a group of donors was to obtain negative control serum samples for an on-going antigen development assay in diagnosis of amoebic liver abscess. Out of 200 samples, 125 (62.5%) were negative, whereas 44 (21.5%) had IHA titer of less than 1:128 and 31 (16.0%) of the samples had significant IHA titers of 1:128 or more, in which 2 serum samples gave titers of 1:4096.