Bwanga Freddie, Disqué Claudia, Lorenz Michael G, Allerheiligen Vera, Worodria William, Luyombya Allan, Najjingo Irene, Weizenegger Michael
Makerere University College of Health Sciences, Kampala, Uganda.
MBN Clinical Laboratories, Kampala, Uganda.
BMC Infect Dis. 2015 Feb 6;15:48. doi: 10.1186/s12879-015-0785-3.
Blood stream tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB) is common among HIV-positive patients, turning rapidly fatal unless detected and treated promptly. Blood culture is currently the standard test for the detection of MTB in whole blood but results take weeks; patients deteriorate markedly and often die before a diagnosis of blood stream TB is made. Rapid molecular tests on whole blood, with potential for same day diagnosis of blood stream TB usually show low sensitivity due to the problem of insufficient MTB DNA template when extraction is performed directly on low blood volumes. This study assessed the influence of blood volume on the sensitivity of a HyBeacon PCR assay-the FluoroType MTB (Hain Lifescience, Nehren, Germany) on direct detection of MTB in whole blood.
Prospective recruitment of HIV-positive patients with clinical suspicion of blood stream TB but not on anti-TB or HIV drug treatment was done. Venous blood samples were collected and DNA extracted using the MolYsis (Molzym, Bremen, Germany) methods; for study A, from duplicate 1 ml (42 patients) and for study B (31 patients) from 9 ml EDTA blood samples. The FluoroType MTB PCR assay targeting an IS6110 sequence was performed and results compared with blood culture.
The diagnostic sensitivity and specificity of the FluoroType MTB PCR in study A was 33% and 97%, respectively. Corresponding values in study B were 71% and 96%, respectively. In both studies, one case each of blood culture-negative blood stream TB was detected with the FluoroType MTB PCR assay. The median time to positivity of blood culture was 20.1 (range 12-32) for study A and 19.9 days (range 15-30) for study B.
Larger blood volumes (9 ml) improved and gave acceptable sensitivity of direct PCR diagnosis of blood stream TB.
由结核分枝杆菌（MTB）引起的血流感染性结核病在HIV阳性患者中很常见，除非及时发现并治疗，否则会迅速致命。血培养是目前检测全血中MTB的标准方法，但结果需要数周时间；患者病情会显著恶化，常在血流感染性结核病确诊前死亡。对全血进行快速分子检测，虽有可能在同一天诊断出血流感染性结核病，但由于直接对少量血液进行提取时MTB DNA模板不足的问题，其敏感性通常较低。本研究评估了血样体积对HyBeacon PCR检测法——FluoroType MTB（德国内伦市海因生命科学公司）直接检测全血中MTB敏感性的影响。
前瞻性招募临床怀疑患有血流感染性结核病但未接受抗结核或HIV药物治疗的HIV阳性患者。采集静脉血样本，使用MolYsis（德国不来梅市Molzym公司）方法提取DNA；研究A中，从1毫升的双份血样（42例患者）中提取，研究B（31例患者）则从9毫升乙二胺四乙酸（EDTA）血样中提取。进行针对IS6110序列的FluoroType MTB PCR检测，并将结果与血培养结果进行比较。
研究A中FluoroType MTB PCR的诊断敏感性和特异性分别为33%和97%。研究B中的相应值分别为71%和96%。在两项研究中，通过FluoroType MTB PCR检测均各发现1例血培养阴性的血流感染性结核病病例。研究A血培养阳性的中位时间为20.1天（范围12 - 32天），研究B为19.9天（范围15 - 30天）。
更大的血样体积（9毫升）提高了血流感染性结核病直接PCR诊断的敏感性，并使其达到了可接受的水平。
