Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients
Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients
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DOI：
10.1099/jmm.0.016261-0
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发表时间：
2010-06-01
影响因子：
3
通讯作者：
Pereira-Chioccola, Vera L.
中科院分区：
文献类型：
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作者：
Mesquita, Rafael T.;Ziegler, Angela P.;Pereira-Chioccola, Vera L.
脑弓形虫病是巴西艾滋病患者最常见的脑部肿块病变，尽管可以免费获得高效抗逆转录病毒治疗，但仍导致高死亡率和发病率。基于常规PCR（cnPCR）或实时定量PCR（qrtPCR）的分子诊断已成为确定性诊断不可或缺的方法。我们在这里报告的qrtPCR与血液和脑脊液（CSF）样本在巴西的艾滋病患者的评价。本前瞻性研究进行了2年，分析了从149例经临床和放射学确诊的艾滋病患者（98例血液和51例CSF样本）中提取的DNA样本。实验室诊断包括cnPCR（使用B22/B23引物组）和间接免疫荧光（IF）。对于qrtPCR，基于所述基因并使用6-羧基荧光素染料标记的TaqMan MGB（小沟结合剂）探针同时设计两个引物组，一个是Bug，其扩增来自B1基因的序列，另一个是RETg，其扩增529 bp序列的PCR产物。在33.6%（50）的患者中观察到总体cnPCR和qrtPCR结果为阳性结果。cnPCR（B22/B23）的敏感性为98%，qrtPCR（B1 Tg和RETg）的敏感性分别为86%和98%。66.4%的患者出现阴性反应。cnPCR和qrtPCR（B1 Tg）的特异性为97%。RETg为88.8%。这些数据表明，RETg PCR是高度敏感的，因为它扩增了具有许多拷贝的重复区;而B1 Tg PCR特异性好，但敏感性低。在20例患者中，同时采集血液和CSF，血清学和临床诊断，以便更好地了解实验室和临床诊断的不同情况。对于9例确诊为脑弓形体病的患者，观察到4种情况：（i）和（ii）CSF分子诊断阴性，血液阳性，血清和CSF的IF滴度可变（阴性或阳性），（iii）CSF分子诊断阳性，血液阴性，以及（iv）两种样品的分子诊断均阳性。在后两种情况下，血清和CSF中的IF滴度通常是可变的。尽管血清和CSF中的IF滴度是可变的，但11名患者中显示了其他机会性感染，所有患者对两种样品的分子诊断均为阴性。qrtPCR允许快速鉴定患者样品中的弓形虫DNA;在少数情况下，与cnPCR发生差异。
Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe One was Bug, which amplified a sequence from the B1 gene The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were positive results were observed in 33.6% (50) patients The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66 4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg). and 88.8% for RETg These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers However, B1Tg PCR had good specificity, but lower sensitivity Among the patients, 20 had blood and CSF collected simultaneously Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive), (iii) positive molecular diagnosis with CSF and negative with blood, and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.