小儿隐孢子虫病的实验诊断研究

Laboratory study on diagnosis of cryptosporidiosis in children with diarrhea

目的 探讨不同检测方法对小儿腹泻粪便中隐孢子虫卵囊 (CSO)的诊断效果。方法对 6 49例急性腹泻患儿的粪便采用 7种方法检测CSO :(1)番红 美兰染色法 (SMB) ;(2 )改良抗酸染色法 (MAF) ;(3)蔗糖漂浮 (SSF)后SMB染色法 ;(4)PBS 乙醚 (PBS E)离心沉淀后MAF染色法 ;(5 )聚蔗糖 泛影酸钠 (FSD)纯化后扫描电镜法 (SEM) ;(6 )FSD或Percoll纯化后免疫组化法 (ABC) ;(7)Percoll纯化后间接免疫荧光法 (IFA)。对 6 49例患儿的新鲜粪便标本同时采用前 4种方法检测 ,而对患儿的经预处理低温存放的 710份标本 (已知阳性 86份 ,已知阴性 6 2 4份 )同时采用 7种方法检测。结果 共检出隐孢子虫腹泻病 2 6例 ,阳检率 4.0 %。SMB和MAF(SMB +MAF)方法的敏感性、特异性分别为80 .2 %和 94.1%、6 1.6 %和 98.4% (P <0 .0 1) ,它们的检测准确性分别为 92 .4%和 93.9% (P >0 .0 5 ) ,SSF +SMB、PBS E法 +MAF法及ABC法的敏感性、特异性、准确性均为 10 0 % ,与SMB、MAF法相比较均有非常显著性意义 (P <0 .0 1)。结论 粪便直接涂片SMB法阳检率高 ,假阳性也高 ,适宜筛查 ;MAF法与之相反 ,漏诊率高 ;ABC法与金标准IFA法检测效果与IFA法一样 ,但方法繁琐造价高 ;SSF或PBS E法浓集CSO后SMB、MAF法的检测效果与IFA法相似 ,且经济、?

Objective To evaluate the diagostic effects, sensitivity, specificity and accuracy of 7 different methods in detecting cryptosporidium oocysts (CSO) in fecal specimens of children with diarrhea. Methods Seven microscopy-based CSO detection methods were applied to unconcentrated and concentrated fecal smears from 649 children with acute diarrhea. The seven methods were as follows: (1) A direct hot safranin-methylene blue (SMB) stain method, (2)a direct modified acid-fast (MAF) stain method, (3)a modified Sheather′s sucrose flotation (SSF) plus SMB stain method, (4) a PBS-ether concentration (PBS-E) plus MAF stain, (5) a combination of Ficoll-sodium diatrizoate (FSD) discontinuous density gradient and observation under scanning electron microscope (SEM), (6) FSD or Percoll purifying CSO method plus an avidin-biotin-peroxidase complex (ABC) technique, and (7) a monoclonal antibody-based indirect immunofluorescence assay (IFA). The first 4 methods listed above were simultaneously used to examine CSO in fresh stool samples from 649 children with diarrhea. All seven methods were used in 710 previously collected, pretreated, stored (at 2-8℃) specimens (86 known positives, 624 known negatives) from 649 children with diarrhea. Results A total of 649 diarrhea cases were examined for fecal CSO, which were identified in 26 cases (4.0%). The sensitivity, specificity and accuracy of the SMB stain and the MAF (or SMB+MAF) method for the fecal CSO were 80.2% and 94.1%, 61.6% and 98.4% ( P < 0.01), 92.4% and 93.9% ( P >0.05), respectively. The sensitivity, specificity and accuracy of the SSF+SMB, PBS-E+MAF, ABC methods were all 100%, which were statistically significant ( P <0.01 for all) as compared to those of SMB, MAF or SMB+MAF methods. Conclusions The direct SMB staining of the unconcentrated fecal smears showed a higher positive rate than the one of the direct MAF stain, but its mis-diagnosis rate was higher as well. This test may be suitable for screening. The MAF stain had a high false-negative rate. The detective effect of ABC was the same as IFA with monoclonal antibodies, but the performing process was complicated, manifold and the reagents are expensive. The diagnostic effects of SSF+SMB and PBS-E plus MAF methods were similar, 100% in sensitivity, specificity or accuracy, to one of the gold standard methods-IFA. These two methods were easy in visual recognition, inexpensive, simple and convenient, therefore might be the ideal methods for the detection of fecal CSO.