Value of fluorescence staining in histopathological diagnosis of Acanthamoeba keratitis / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the application value of fluorescent staining technique in the detection of amoebic pathogens in corneal tissue biopsy, and to apply fluorescent staining technique in the histopathological diagnosis of Acanthamoeba keratitis(AK), comparing the results with those of hemotoxyiln-eosin staining(HE staining)and periodic acid-schiff staining(PAS staining), and analyzing the sensitivity and specificity of these three staining methods.<p>METHODS:Specimens of infected corneal tissue were collected from 74 cases(75 eyes), and then they were divided into an AK group and a non-Acanthamoeba keratitis(NAK)group based on the results of corneal scraping, culture and histopathological diagnosis. The tissues of consecutive sections were stained with HE staining, PAS staining and fluorescence respectively, and the sensitivity and specificity of the three staining methods for the diagnosis of AK were analyzed. Area under the curve(AUC)was calculated using the receiver operating characteristic(ROC)curve. Further analysis was performed to count the number of Acanthamoeba pathogens found by the three staining methods under the same magnification field of view at the same site, and to clarify the diagnostic value of fluorescent staining technique for AK.<p>RESULTS: The sensitivity of HE staining was 69%(27/39)with a specificity of 92%; the sensitivity of PAS staining was 62%(24/39)with a specificity of 97%, and the sensitivity of fluorescent staining was 95%(37/39)with a specificity of 97%. There were differences in the sensitivity of the three staining methods for the diagnosis of AK(χ2=19.857, <i>P</i><0.001), and pairwise comparison revealed that the differences between HE staining and fluorescent staining, PAS staining and fluorescent staining for the diagnosis of AK were statistically significant(<i>P</i>=0.003,<0.001), while the difference in sensitivity between HE staining and PAS staining for the diagnosis of AK was not statistically significant(<i>P</i>=0.978). The maximum AUC was 0.960 for fluorescence staining, followed by 0.804 for HE staining and 0.794 for PAS staining, respectively. The median number of amoeba cysts detected by HE staining, PAS staining and fluorescent staining at the same site under the same magnification field of view was 4(0, 11), 2(0, 9)and 12(3, 33), respectively(χ2=56.561, <i>P</i><0.001). Pairwise comparison revealed that the differences in the number of amoeba cysts found by HE staining and fluorescence staining, PAS staining and fluorescence staining were statistically significant(<i>P</i><0.001), while the difference in the number of amoeba cysts found by HE staining and PAS staining was not statistically significant(<i>P</i>=0.210). Fluorescently stained histopathological sections make it easier to identify amoebic pathogens.<p>CONCLUSION:Fluorescent staining technique is more sensitive to histopathological diagnosis of AK than HE staining and PAS staining, which can significantly improve the positive rate of detection of amoebic pathogens.

Identification and glycobiological characterization of circulating immune complexes in patients with visceral leishmaniasis and post kala azar dermal leishmaniasis.

Here, we investigated the quantitative and qualitative differences in antibody classes and subclasses in serum immune complexes (ICs) of Visceral Leishmaniasis (VL), Post Kala-azar Dermal Leishmaniasis (PKDL) and different cross reactive diseases like Malaria, Leprosy, Vitiligo as compared to control subjects. IC levels were measured through a newly developed PEG ELISA, using L. donovani promastigote membrane antigen coated plate. Antibody classes and subclasses were identified using polyspecific sera and monoclonal antibodies, respectively. ICs were purified using polyethylene glycol (PEG) precipitation. Conditional logistic regression showed an association between IgG1-containing ICs and increased risk of PKDL (OR=75, P <0.05) and an association of IgG-containing ICs with VL (OR=621, P=0.001). PEG ELISA demonstrated almost 13-15 fold higher IgG containing ICs titers in VL as compared to control (P <0.001). The assay further established a significant (P <0.05) difference in the IgG containing ICs titers between VL and PKDL. The isolated ICs were further analyzed by subjecting them to one-dimensional PAGE and subsequently stained with combination of periodic acid schiff (PAS) with silver. A differential banding pattern between VL and PKDL was obtained. Four distinct bands with carbohydrate rich glycoconjugates were identified in PKDL ICs, which were absent in VL and control group. It suggests the scope for developing a novel differential diagnostic assay.

Application of special staining techniques in pathological diagnosis of fungi infections in HIV/AIDS patients / 中华临床感染病杂志

Objective To apply special staining techniques in pathological diagnosis of fungal infections in HIV/AIDS patients.Methods Pathological data of 20 HIV/AIDS patients complicated with fungi infections in Shanghai Public Health Clinical Center during February 2010 and November 2013 were retrospectively analyzed.Tissue specimens were stained with hematoxylin and eosin (HE),Periodic acidSchiff (PAS) and methenamine silver nitrate (MSN),and the sections were observed under optical microscope.Results Among 20 HIV/AIDS patients complicated with fungi infections,2 were infected with pulmonary cryptococcosis; 3 were penicillium marneffei infections in skin,lung and abdominal mesenteric lymph nodes; 5 were histoplasma capsulatum infections in epiglottis,neck lymph nodes,oral cavity,abdominal cavity and skin; 4 were aspergillus infections in maxillary sinus,lung and vocal cords,and 3 of them were combined with tuberculous lesions; 6 were candida albicans infections in liver,pharynx,esophagus and stomach.In tissues stained with HE the infiltration of inflammatory cells,granuloma formation and coagulative necrosis were observed,and the shape of fungi needed careful observation to avoid missed diagnosis and misdiagnosis.In tissues stained with PAS,fungal spores and pseudohypha were presented in bright amaranth,and cell nucleus was in purple-blue.In tissues stained with MSN,fungal spores and pseudohypha were identified clearly in brown-black.Conclusion HE plus PAS and MSN staining will help pathological diagnosis of fungi infection.