使用免疫磁分离和抗菌去污技术增强呼吸道样本中军团菌的培养。,Journal of Cli
使用免疫磁分离和抗菌去污技术增强呼吸道样本中军团菌的培养。
Journal of Clinical Microbiology
(
IF
6.1
)
Pub Date : 2020-10-21
, DOI:
10.1128/jcm.01218-20
Ali Mohammadi
1
,
Stephen T Chambers
2,
3
,
Amy Scott-Thomas
1
,
John G Lewis
4
,
Trevor Anderson
5
,
Ros Podmore
5
,
Jonathan Williman
6
,
David Murdoch
1,
4
,
Sandy Slow
1
Affiliation
Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand.
Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand
Department of Infectious Diseases, Christchurch Hospital, Christchurch, New Zealand.
Department of Biochemistry, Canterbury Health Laboratories, Christchurch, New Zealand.
Department of Microbiology, Canterbury Health Laboratories, Christchurch, New Zealand.
Department of Population Health, University of Otago, Christchurch, New Zealand.
长生军团菌是新西兰社区获得性肺炎患者中最常见的军团菌种。在培养物中分离生物是诊断军团菌疾病的金标准，居室保洁13825404095但与定量PCR（qPCR）相比，其敏感性较差（40％）。我们已经开发了使用甘氨酸，万古霉素，多粘菌素，放线菌酮和（GVPC）用免疫磁性分离（IMS）用于培养选择性净化过程L. longbeachae。从新西兰白兔生产长双歧杆菌特异的多克隆抗体，并将其与甲苯磺酰基活化的磁珠偶联。存储L. longbeachae从-80°C的存储中检索qPCR阳性呼吸道样品进行测试。将一份测试样品与GVPC和抗体珠复合物混合，分离，洗涤并在改良的Wadowsky和Yee琼脂（MWY）琼脂上培养。在MWY琼脂上孵育之前，将另一部分暴露于HCl-KCl酸性缓冲液（pH 2.2）。qPCR使用对长滩乳杆菌基因组ITS（内部转录间隔区）区域具有特异性的探针。酸洗后10/53（19％）样品中培养阳性，GVPC-IMS后培养26/53（49％）样品中培养阳性（P = 0.001）。污染物的生长很少。酸洗后培养阳性样品的平均qPCR阈值循环值比培养阴性样品低（平均值为29.9比34.8；差异为4.9； 95％置信区间[CI]为±2.9；平均值为2.9。P = 0.001），但在GVPC-IMS之后没有发生（平均值为33.0对34.7;差异为1.7; 95％CI为±2.48; P = 0.16）。培养的用于灵敏度L. longbeachae在呼吸道标本可以通过使用GVPC-IMS，而不是用于去污酸洗涤得到改善，但这应该在的新鲜标本的前瞻性研究来确认。
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Enhancement of culture of Legionella longbeachae from respiratory samples using immunomagnetic separation and antimicrobial decontamination.
Legionella longbeachae is the commonest Legionella species identified in patients with community-acquired pneumonia in New Zealand. Isolation of the organism on culture is the gold standard for the diagnosis of Legionnaires disease, but it has poor sensitivity (40%) compared with quantitative PCR (qPCR). We have developed a selective decontamination process using glycine, vancomycin, polymyxin, and cycloheximide (GVPC) with immunomagnetic separation (IMS) for culturing L. longbeachae. A polyclonal antibody specific for L. longbeachae was produced from New Zealand White rabbits and coupled to tosyl-activated magnetic beads. Stored L. longbeachae qPCR-positive respiratory samples were retrieved from −80°C storage for testing. One portion of test samples was mixed with GVPC and the antibody bead complex, separated, washed, and cultured on modified Wadowsky and Yee agar (MWY) agar. Another portion was exposed to HCl-KCl acidic buffer (pH 2.2) before incubation on MWY agar. qPCR used probes specific for the ITS (internal transcribed spacer) region of the L. longbeachae genome. Cultures were positive in 10/53 (19%) samples after acid wash and 26/53 (49%) after GVPC-IMS (P = 0.001). Growth of contaminants was rare. The mean qPCR threshold cycle values were lower in culture-positive samples after acid wash than in the culture-negative samples (mean, 29.9 versus 34.8; difference, 4.9; 95% confidence interval [CI], ±2.9; P = 0.001) but not after GVPC-IMS (mean, 33.0 versus 34.7; difference, 1.7; 95% CI, ±2.48; P = 0.16). The sensitivity of culture for L. longbeachae in respiratory specimens may be improved by using GVPC-IMS rather than acid wash for decontamination, but this should be confirmed in a prospective study of fresh specimens.