Fauville-Dufaux M, Waelbroeck A, De Mol P, Vanfleteren B, Levy J, Debusschere P, Farber C M
Laboratory of Tuberculosis and Mycobacteria, Institut Pasteur-Bruxelles, Brus
Eur J Pediatr. 1996 Feb;155(2):106-11. doi: 10.1007/BF02075761.
The purpose of the study was to evaluate the contribution of polymerase chain reaction (PCR) to the diagnosis of tuberculous infection in children. Two different PCR techniques were compared to the standard bacteriological methods for the detection of Mycobacterium tuberculosis in 157 specimens obtained from the respiratory system of 51 children. Patients were classified in three groups: 12 patients with active disease (57 specimens), 12 patients with silent tuberculous infection (23 specimens) and 27 patients without tuberculosis (77 specimens). One PCR method (PCR/Ag85) used amplification of a fragment of the genes coding for the mycobacterial antigen 85 followed by hybridization of a probe specific for M. tuberculosis on the Southern blot of amplified DNA. The other PCR technique was a nested PCR (NPCR) using double amplification of a fragment of the insertion element IS6110 only present in the M. tuberculosis genome. The sensitivities of the different techniques, compared to the clinical diagnosis, were 7.0% for acid fast staining, 22.8% for culture, 24.6% for PCR/Ag85 and 44.9% for NPCR in active disease, 4.3% for culture, 8.7% for PCR/Ag85 and 28.6% for NPCR in silent tuberculous infection. The specificities were 100% for culture, 94.8% for PCR/Ag85 and 87.9% for NPCR. Among the 12 children clinically considered as having active tuberculosis, 1 had smear positive samples, 4 had at least one positive culture, 7 at least one positive PCR/Ag85 and 9 at least one NPCR positive sample. Among the 12 children having silent tuberculous infection, none had positive smears, 1 had one positive culture, 2 had at least one positive PCR/Ag85 and 5 at least one NPCR positive sample.
Our study suggests that both PCR techniques, and especially NPCR, are able to detect M. tuberculosis DNA in specimens containing few micro-organisms. PCR methods are more sensitive than culture and the results are available more quickly. Testing multiple samples from the same individual increased the sensitivity. In view of occasional false-positive results, cultures remain the gold standard to establish definitive diagnosis of primary tuberculous infection in children.
本研究的目的是评估聚合酶链反应（PCR）在儿童结核感染诊断中的作用。将两种不同的PCR技术与标准细菌学方法进行比较，以检测从51名儿童呼吸系统获取的157份标本中的结核分枝杆菌。患者分为三组：12例活动性疾病患者（57份标本）、12例潜伏结核感染患者（23份标本）和27例无结核病患者（77份标本）。一种PCR方法（PCR/Ag85）是扩增编码分枝杆菌抗原85的基因片段，然后在扩增DNA的Southern印迹上用结核分枝杆菌特异性探针进行杂交。另一种PCR技术是巢式PCR（NPCR），仅对结核分枝杆菌基因组中存在的插入元件IS6110的片段进行双重扩增。与临床诊断相比，不同技术的敏感性在活动性疾病中，抗酸染色为7.0%，培养为22.8%，PCR/Ag85为24.6%，NPCR为44.9%；在潜伏结核感染中，培养为4.3%，PCR/Ag85为8.7%，NPCR为28.6%。培养的特异性为100%，PCR/Ag85为94.8%，NPCR为87.9%。在临床上被认为患有活动性结核病的12名儿童中，1名涂片阳性，4名至少有一次培养阳性，7名至少有一次PCR/Ag85阳性，9名至少有一次NPCR阳性。在12名潜伏结核感染儿童中，无人涂片阳性，1名培养阳性，2名至少有一次PCR/Ag85阳性，5名至少有一次NPCR阳性。
我们的研究表明，两种PCR技术，尤其是NPCR，能够在含有少量微生物的标本中检测到结核分枝杆菌DNA。PCR方法比培养更敏感，结果获取更快。对同一个体检测多个样本可提高敏感性。鉴于偶尔出现假阳性结果，培养仍然是确立儿童原发性结核感染明确诊断的金标准。
