Application of multi-channel molecular beacon polymerase chain reaction in rapid diagnosis of neonatal invasive Candida infection

doi:10.3877/cma.j.issn.1674-1358.2019.05.007

Abstract

Abstract:

Objective

To preliminarily establish a multi-channel molecular beacon polymerase chain reaction (MQ-PCR) method for early detection of common Candida pathogens associated with invasive fungal infections (IFI) in newborns, and to explore the differences of Candidas detection in different parts of neonates with IFI.

Methods

Throat swabs, deep sputum and blood samples from 34 neonates suspected of invasive fungal infection in Neonatal Intensive Care Unit (NICU), Beijing Obstetrics and Gynecology Hospital, Capital Medical University, from January 2018 to April 2019 were collected. Blood culture and strain identification were performed routinely, and Candidas were also detected by MQ-PCR system in three types of samples. The positive detection rate and detection period of the two methods were compared, respectively.

Results

The lower detection limits of the MQ-PCR system for Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis were all 50 CFU/ml, and the linear detection ranges were all 5 × 10¹-1 × 10⁵ CFU/ml. Bacteria detected by MQ-PCR system did not cross-react with 14 other common pathogenic bacteria or fungi with high homology DNA sequence. IFI was identified in 9 cases (26.47%) among the 34 infants by positive Candidas in blood culture. Meanwhile, the same blood sample and the patient’s own (deep sputum and pharyngeal swab) samples from different parts of the above 9 children with IFI were also positive for Candidas by MQ-PCR, and the detected strains were identical with blood culture. The positive detection rate of Candida in blood samples by the two methods was basically the same (χ² = 0.5, P = 0.5), which were 26.5% (9/34) by the culture method and 32.4% (11/34) by MQ-PCR. Compared with the positive detection rate of Candida in blood samples by blood culture (26.5%), there was no significant difference in different parts of the samples by MQ-PCR (all P > 0.05), which were 41.2% (14/34) in pharyngeal swabs, 32.4% (11/34) in deep sputum, and 32.4% (11/34) in blood. Candida detection period of MQ-PCR in blood [3.5 (3.2, 3.9); Z =-2.7, P = 0.008], deep sputum [3.1 (2.9, 3.2); Z =-2.5, P = 0.013], swabs [2.7 (2.5, 2.9); Z =-2.7, P = 0.008] were significantly shorter than that of Candida blood culture [94.0 (78, 105.8)].

Conclusions

Compared with blood culture, MQ-PCR had the advantages of high coincidence rate and early detection when used to detect common Candidas pathogens in multi-types of specimens from neonatal IFI patients. MQ-PCR was helpful for early diagnosis of bloodstream infection and dynamic monitoring of Candida colonization in neonates.

Key words: Newborns, Invasive Candidas infection, Multi-channel molecular beacon polymerase chain reaction, Early-warning

Yu Zhang, Huihui Zeng, Chunling Wen, Xiaoyi Cai, Na Zhao, Xingwang Li. Application of multi-channel molecular beacon polymerase chain reaction in rapid diagnosis of neonatal invasive Candida infection[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2019, 13(05): 382-388.

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Figures/Tables 5

References 21

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菌株类型	引物序列F（5'→3'）	引物序列R（5'→3'）	探针序列（5'→3'）
白色念珠菌	GGCGCTCAAGCCTGCTACT	ACGCCTGCCTACTCGTGAA	ATACTTCACCGTGATTGCTGTTTTGACGC
近平滑念珠菌	CATTTGCTCAATTGTATGCTGATTT	CGCAGCATCACGTTTCCA	CCCCAATCAATTTTGTTTTCCCACACTTG
光滑念珠菌	CGAGAACAACTCCGATTCTATAACC	TGGACTCAGAATCAGCATTTTCA	CCAATGCGACCAACGAAAAACACATG
热带念珠菌	ATAACAGGTCTGTGATGCCCTTAGA	CGCTGGCTCCGTCAGTGTA	TTCTGGGCCGCACGCGC

菌株类型	引物序列F（5'→3'）	引物序列R（5'→3'）	探针序列（5'→3'）
白色念珠菌	GGCGCTCAAGCCTGCTACT	ACGCCTGCCTACTCGTGAA	ATACTTCACCGTGATTGCTGTTTTGACGC
近平滑念珠菌	CATTTGCTCAATTGTATGCTGATTT	CGCAGCATCACGTTTCCA	CCCCAATCAATTTTGTTTTCCCACACTTG
光滑念珠菌	CGAGAACAACTCCGATTCTATAACC	TGGACTCAGAATCAGCATTTTCA	CCAATGCGACCAACGAAAAACACATG
热带念珠菌	ATAACAGGTCTGTGATGCCCTTAGA	CGCTGGCTCCGTCAGTGTA	TTCTGGGCCGCACGCGC

患儿编号	胎龄（周）	感染发生日龄	咽拭子PCR	深部痰液PCR	血液PCR	血培养
1	27	23	白色念珠菌	白色念珠菌	白色念珠菌	白色念珠菌
2	29	35	白色念珠菌	白色念珠菌	白色念珠菌	白色念珠菌
3	26	17	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
4	30	19	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
5	31	27	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
6	26	9	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
7	27	29	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
8	33	17	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
9	29	13	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
10	28	31	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	—
11	27	11	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	屎肠球菌
12	36	15	白色念珠菌	—	—	草绿链球菌
13	40	10	白色念珠菌	—	—	—
14	39	8	白色念珠菌	—	—	—
15	31	20	—	—	—	屎肠球菌
16	29	35	—	—	—	表皮葡萄球菌
17	27	33	—	—	—	绿脓杆菌
18	28	38	—	—	—	鲍曼不动杆菌
19	27	47	—	—	—	屎肠球菌
20	35	12	—	—	—	大肠埃希菌
21	27	33	—	—	—	肺炎克雷伯菌
22	31	51	—	—	—	鲍曼不动杆菌
23	29	23	—	—	—	屎肠球菌
24	40	17	—	—	—	表皮葡萄球菌
25	31	20	—	—	—	屎肠球菌
26	30	30	—	—	—	金黄色葡萄球菌
27	28	26	—	—	—	肺炎克雷伯菌
28	30	21	—	—	—	表皮葡萄球菌
29	33	18	—	—	—	表皮葡萄球菌
30	38	13	—	—	—	草绿链球菌
31	36	9	—	—	—	屎肠球菌
32	31	23	—	—	—	—
33	34	14	—	—	—	—
34	29	7	—	—	—	—

患儿编号	胎龄（周）	感染发生日龄	咽拭子PCR	深部痰液PCR	血液PCR	血培养
1	27	23	白色念珠菌	白色念珠菌	白色念珠菌	白色念珠菌
2	29	35	白色念珠菌	白色念珠菌	白色念珠菌	白色念珠菌
3	26	17	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
4	30	19	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
5	31	27	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
6	26	9	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
7	27	29	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
8	33	17	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
9	29	13	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌
10	28	31	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	—
11	27	11	近平滑念珠菌	近平滑念珠菌	近平滑念珠菌	屎肠球菌
12	36	15	白色念珠菌	—	—	草绿链球菌
13	40	10	白色念珠菌	—	—	—
14	39	8	白色念珠菌	—	—	—
15	31	20	—	—	—	屎肠球菌
16	29	35	—	—	—	表皮葡萄球菌
17	27	33	—	—	—	绿脓杆菌
18	28	38	—	—	—	鲍曼不动杆菌
19	27	47	—	—	—	屎肠球菌
20	35	12	—	—	—	大肠埃希菌
21	27	33	—	—	—	肺炎克雷伯菌
22	31	51	—	—	—	鲍曼不动杆菌
23	29	23	—	—	—	屎肠球菌
24	40	17	—	—	—	表皮葡萄球菌
25	31	20	—	—	—	屎肠球菌
26	30	30	—	—	—	金黄色葡萄球菌
27	28	26	—	—	—	肺炎克雷伯菌
28	30	21	—	—	—	表皮葡萄球菌
29	33	18	—	—	—	表皮葡萄球菌
30	38	13	—	—	—	草绿链球菌
31	36	9	—	—	—	屎肠球菌
32	31	23	—	—	—	—
33	34	14	—	—	—	—
34	29	7	—	—	—	—

念珠菌	血培养	MQ-PCR检测
念珠菌	血培养	血液	深部痰液	咽拭子
阳性	9（26.47）	11（32.35）^a	11（32.35）^a	14（41.18）^a
阴性	25（73.53）	23（67.65）	23（67.65）	20（58.82）

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