2020—2021年上海市7例输入性疟疾病例误判原因分析

doi:10.12140/j.issn.1000-7423.2023.01.010

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (1): 68-74.doi: 10.12140/j.issn.1000-7423.2023.01.010

2020—2021年上海市7例输入性疟疾病例误判原因分析

张耀光(), 江莉^*(), 王真瑜, 朱民, 朱倩, 马晓疆, 余晴, 陈健

上海市疾病预防控制中心，上海 200336

收稿日期:2022-06-10 修回日期:2022-10-11 出版日期:2023-02-28 发布日期:2023-02-27
通讯作者: * 江莉（1964-），女，博士，主任技师，主要从事寄生虫病检测研究。E-mail：jiangli@scdc.sh.cn
作者简介:张耀光（1985-），男，本科，副主任技师，从事寄生虫病研究。E-mail：zhangyaoguang@scdc.sh.cn
基金资助:
上海市“科技创新行动计划”项目(20DZ2200300);上海市第五轮公共卫生体系建设三年行动计划重点学科项目(GWV-10.1-XK13)

Analysis of the causes of misdiagnosis of seven imported malaria cases in Shanghai from 2020 to 2021

ZHANG Yaoguang(), JIANG Li^*(), WANG Zhenyu, ZHU Min, ZHU Qian, MA Xiaojiang, YU Qing, Chen Jian

Shanghai Municipal Center for Disease Control and Prevention, Shanghai 200336, China

Received:2022-06-10 Revised:2022-10-11 Online:2023-02-28 Published:2023-02-27
Contact: * E-mail: jiangli@scdc.sh.cn
Supported by:
Shanghai Scientific and Technological Innovation Action Plan(20DZ2200300);Fifth Round Three-Year Action Plan on Key Subject of Shanghai Public Health System Construction(GWV-10.1-XK13)

摘要/Abstract

摘要：

目的通过实验室检测结果综合分析输入性疟疾病例样品的误判原因，为减少误判发生提供参考。方法收集2020—2021年各区疾控中心上报的输入性疟疾病例样品共112份[含血涂片、抗凝血、区疾控中心提取的疟原虫DNA（为区疾控组DNA）等]，由市级疟疾诊断参比实验室进行复核，分别进行显微镜检查、提取疟原虫基因组DNA（为市疾控组DNA）和多重PCR法检测。对其中镜检为阳性且区疾控中心报告荧光定量PCR（qPCR）结果为阴性的病例样品，分别加做qPCR检测，绘制扩增曲线，测量循环阈值（Ct），比较两组DNA的浓度和纯度。结果区疾控中心上报镜检样品112份，其中110份进行核酸检测；108份为疟原虫阳性样品，4份为阴性样品。经市疾控中心复核，镜检结果一致率为93.8%（105/112），其中阳性一致率为94.1%（96/102），阴性一致率为9/10；共筛选出7份输入性疟疾病例误判，其中定性错误3份（2份恶性疟误判为阴性，1份阴性误判为恶性疟），定种错误4份（1份三日疟误判为卵形疟，1份三日疟无法分型，2份卵形疟误判为间日疟）。经市疾控中心复核，核酸检测一致率为96.4%（106/110），其中阳性一致率为96.2%（102/106），阴性一致率为4/4。7例误判病例的区疾控组DNA多重PCR未扩增，市疾控组正常扩增；市、区疾控组DNA在qPCR检测中均正常扩增，市疾控组DNA对应样品的Ct值更低，扩增曲线更早进入指数增长期。区疾控组DNA浓度均大于市疾控组，而市疾控组DNA纯度更高。结论相关区疾控中心需提高疟原虫镜检能力，并选择合适的DNA提取试剂盒和qPCR试剂盒用于疟原虫核酸检测。

关键词: 输入性疟疾, 显微镜检查, 核酸检测, DNA提取, 误判

Abstract:

Objective Comprehensive analysis of laboratory test results of the samples from imported malaria cases to determine the causes of misdiagnosis, to provide reference for reducing the occurrence of misdiagnosis. Methods A total of 112 samples of imported malaria cases reported by each district centers for disease control and prevention (CDCs) from 2020 to 2021 were collected, including blood smears, anticoagulated blood, and Plasmodium genomic DNA extracted by district CDCs (district CDC group). The samples were rechecked by the municipal malaria reference laboratories through microscopic examination, genomic DNA extraction (municipal CDC group) and multiplex PCR assay, respectively. Among the samples of microscopic positives, where as real time quantitative PCR (qPCR) negatives reported by the district CDC, additional qPCR was conducted to plot amplification curves, determine the cycle threshold (Ct) values and compare the concentration and purity of DNA between the two groups. Results Microscopic examination was performed for 112 samples and nuclear acid detection were performed for 110 samples by the district CDCs. 108 were positive samples of Plasmodium and 4 were negative samples. The microscopic concordance rate was 93.8% (105/112), as reviewed by the municipal CDC. Among them, the positive concordance rate was 94.1% (96/102) and the negative concordance rate was 9/10. A total of seven imported malaria cases were screened for misdiagnosis. Three samples had qualitative identification mistakes by the district CDCs (two P. falciparum cases were misdiagnosed as negative, and one negative case was misdiagnosed as P. falciparum). Four samples have species identification mistakes, one sample was P. malariae misdiagnosed as P. ovale, one sample was P. malariae misdiagnosed as undefined, and two samples were P. ovale misdiagnosed as P. vivax. The concordance rate of nucleic acid detection was 96.4% (106/110), as reviewed by the municipal CDC, with a positive concordance rate of 96.2% (102/106) and a negative concordance rate of 4/4. In seven misdiagnosed cases, there were no amplifications of multiplex PCR the district CDC group, and normal amplifications in the municipal CDC group. The qPCR detections were amplified normally in both district and municipal CDC groups, and the corresponding samples in the municipal CDC group had lower Ct values and the amplification curve entered the exponential growth phase earlier. The DNA concentrations in the district CDC group were all greater than those in municipal CDC group, while municipal CDC group had higher DNA purity. Conclusion Relevant district CDCs need to improve their microscopic examination capability for Plasmodium, and use appropriate DNA extraction kits and qPCR kits for Plasmodium nucleic acid detection.

Key words: Imported malaria, Microscopic examination, Nucleic acid detection, DNA extraction, Misjudgment

中图分类号:

R531.3

张耀光, 江莉, 王真瑜, 朱民, 朱倩, 马晓疆, 余晴, 陈健. 2020—2021年上海市7例输入性疟疾病例误判原因分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(1): 68-74.

ZHANG Yaoguang, JIANG Li, WANG Zhenyu, ZHU Min, ZHU Qian, MA Xiaojiang, YU Qing, Chen Jian. Analysis of the causes of misdiagnosis of seven imported malaria cases in Shanghai from 2020 to 2021[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(1): 68-74.

图/表 7

表1

表2

图1

表3

7例输入性疟疾病例的血涂片镜检及质量情况

病例编号 Case code	疟原虫特征 The feature of Plasmodium	血涂片质量 Quality of blood smears	原虫密度计数（个/μL） Parasite counting (Parasite/μL)
病例1 Case 1	查见恶性疟环状体，核小而致密，胞质较稀疏 The ring form stage of P. falciparum was found, with small and dense nucleus and sparse cytoplasm	无厚血膜 No thick blood membrane	904^a
病例2 Case 2	未查见疟原虫，血片中可见豪焦小体 Parasites were not found, and Howell - Jolly bodies were found in blood smear	良好 Fine	-
病例3 Case 3	查见三日疟配子体和未成熟裂殖体，被寄生红细胞略缩小 The gametophytes and immature schizonites of P. malariae were found, and the parasitized red blood cells were slightly reduced in size	良好 Fine	1 486
病例4 Case 4	大部分疟原虫未能着色，仅查见疟原虫粉色细胞核，胞浆不可见 Most of the parasites failed to be stained, only the pink nucleus was found, and the cytoplasm was not visible	染色过淡 Too light dyeing	235
病例5 Case 5	查见三日疟小滋养体，原虫形态不典型，被寄生红细胞略缩小；厚血膜中查见“菊花状”裂殖体 It was found that the small trophozoites of P. malariae, the patterns were not typical, and the parasitized red blood cells were slightly reduced. “Chrysanthemum shaped”schizonites were found in thick blood membrane	良好 Fine	114
病例6 Case 6	查见卵形疟大滋养体，被寄生红细胞正常或略涨大，边缘呈彗星状、伞矢状拖尾 P. oval large trophozoites were found, and the parasitized red blood cells were normal or slightly enlarged, with comet-like and umbrella-sagittal	良好 Fine	2 182
病例7 Case 7	查见卵形疟大滋养体，被寄生红细胞正常或略涨大，边缘呈彗星状、伞矢状拖尾 P. oval large trophozoites were found, and the parasitized red blood cells were normal or slightly enlarged, with comet-like and umbrella-sagittal	良好 Fine	6 807

表3

图2

图3

表4

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区 Districts	样品数 Total samples	阳性样品数 Positive samples				阴性样品数 Negative samples
区 Districts	样品数 Total samples	恶性疟原虫 P. falciparum	间日疟原虫 P. vivax	卵形疟原虫 P. ovale	三日疟原虫 P. malariae	阴性样品数 Negative samples
浦东 Pudong	23	18	1	3	0	1
闵行 Minhang	21	20	1	0	0	0
嘉定 Jiading	12	9	1	1	1	0
松江 Songjiang	10	9	0	1	0	0
宝山 Baoshan	8	7	0	0	0	1
青浦 Qingpu	8	5	2	0	0	1
杨浦 Yangpu	5	4	0	1	0	0
金山 Jinshan	4	4	0	0	0	0
静安 Jingan	4	2	0	1	0	1
虹口 Hongkou	4	3	1	0	0	0
黄浦 Huangpu	4	4	0	0	0	0
长宁 Changning	4	4	0	0	0	0
奉贤 Fengxian	2	1	0	0	1	0
普陀 Putuo	2	2	0	0	0	0
崇明 Chongming	1	1	0	0	0	0
徐汇 Xuhui	0	0	0	0	0	0
合计 Total	112	93	6	7	2	4

病例编号 Case code	性别 Gender	年龄 Age	外出国家 Country visited	归国时间 Return time	此次发病时间 Onset time	区级报告结果 Reported results of district CDC		市级复核结果 Rechecked results of municipal CDC		病例最终确认结果 Ultimate outcome
病例编号 Case code	性别 Gender	年龄 Age	外出国家 Country visited	归国时间 Return time	此次发病时间 Onset time	镜检 Microscopic detection	PCR	镜检 Microscopic detection	PCR	病例最终确认结果 Ultimate outcome
病例1 Case 1	男Male	30	几内亚Guinea	2020.8.7	2020.8.8	(-)	-	Pf	Pf	Pf
病例2 Case 2	男Male	64	几内亚Guinea	2020.8.25	2020.8.27	Pf	Pf	(-)	Pf	Pf
病例3 Case 3	男Male	32	刚果布Congo	2021.6.18	2021.7.6	Po	(-)	Pm	Pm	Pm
病例4 Case 4	男Male	31	几内亚Guinea	2021.8.10	2021.8.17	(-)	Pf	Pf	Pf	Pf
病例5 Case 5	男Male	35	几内亚Guinea	2021.9.16	2021.9.30	(+)	(-)	Pm	Pm	Pm
病例6 Case 6	男Male	53	几内亚Guinea	2021.12.7	2021.12.9	Pv	(-)	Po	Po	Po
病例7 Case 7	男Male	37	几内亚Guinea	2021.12.7	2021.12.10	Pv	(-)	Po	Po	Po

病例编号 Case code	DNA浓度/ng·μl^-1 DNA concentration/ng·μl^-1		A₂₆₀/A₂₈₀
病例编号 Case code	市疾控 Municipal CDC	区疾控 District CDC	市疾控 Municipal CDC	区疾控 District CDC
病例3 Case 3	42.48	515.68	1.78	1.00
病例5 Case 5	24.70	568.18	1.69	1.22
病例6 Case 6	7.11	706.97	1.77	1.69
病例7 Case 7	14.38	673.44	1.70	1.50

2020—2021年上海市7例输入性疟疾病例误判原因分析

Analysis of the causes of misdiagnosis of seven imported malaria cases in Shanghai from 2020 to 2021

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