Distéfano Angélica Lidia, Alonso Alicia, Martin Fabián, Pardon Fabián
Department of Virology, Laboratorio de Virosis Congénitas Perinatales y Transmisión Sexual, Instituto Nacional de Microbiología INEI-ANLIS Carlos G, Malbrán, Ministerio de Salud de la Nación, Av, CP 1281, Buenos Aires, Argentina.
BMC Pediatr. 2004 Jun 23;4:11. doi: 10.1186/1471-2431-4-11.
Human cytomegalovirus (CMV) is one of the most commonly found agents of congenital infections. Primary maternal infection is associated with risk of symptomatic congenital diseases, and high morbidity is frequently associated with very low birth weight. Neonates with asymptomatic infection develop various sequelae during infancy. This is the first Argentine study performed in neonates with congenital and postnatal HCMV infection. The purpose of this study was to evaluate the performance of the polymerase chain reaction (PCR) technique with different pairs of primers, to detect cytomegalovirus isolated in tissue cultures and directly in urine and dried blood spot (DBS) specimens. Results were compared with IgM detection.
The study was performed between 1999 and 2001 on routine samples in the Laboratory. A total of 61 urine and 56 serum samples were selected from 61 newborns/infants, 33 patients whose samples were analyzed during the first two to three weeks of life were considered congenital infections; the remaining 28 patients whose samples were taken later than the third week were grouped as perinatal infections, although only in 4 the perinatal transmission of infection was determined unequivocally. Cytomegalovirus diagnosis was made by isolating the virus from urine samples in human foreskin fibroblast cells. Three different primer pairs directed to IE, LA and gB genes were used for the HCMV PCR assay in viral isolates. Subsequently, PCR and nested PCR (nPCR) assays with gB primers were performed directly in urine and in 11 samples of dried blood spot (DBS) on Guthrie Card, these results were then compared with serology.
The main clinical manifestations of the 33 patients with congenital infection were purpura, jaundice, hepatomegaly and anaemia. Three patients presented low birth weight as single symptom, 10, intracranial calcifications, and 2, kidney failure. In the 28 patients grouped as with perinatal infection, anaemia, hepatosplenomegaly and enzymatic alteration were predominant, and 4 patients were HIV positive. The primers used to amplify the gB region had a PCR positivity rate of 100%, whereas those that amplified IE and LA regions had a PCR positivity rate of 54% and 61% respectively, in CMV isolates. Amplification by PCR of urine samples (with no previous DNA extraction), using primers for the gB region, detected 34/61 positive samples. Out of the 33 samples from patients with congenital infection, 24 (73%) were positive. When nPCR was used in these samples, all were positive, whereas in the remaining 28 patients, two negative cases were found. Cytomegalovirus DNA detection in 11 samples was also carried out in DBS: 7 DBS samples were positive and 4 were negative.
Primers directed to the gB fragment region were the best choice for the detection of CMV DNA in positive isolates. In congenital infections, direct PCR in urine was positive in a high percentage (73%) of samples; however, in patients grouped as with perinatal infection only 36% of the cases were positive. With n-PCR, total sample positivity reached 97%. PCR technique performed in DBS allowed identifying congenital infection in four patients and to be confirmed in 3. These results show the value of nPCR for the detection of all cases of CMV infection. The assay offers the advantage that it may be performed within the normal working day and provides reliable results in a much shorter time frame than that required for either traditional tissue culture or the shell-viral assay.
人巨细胞病毒(CMV)是先天性感染中最常见的病原体之一。孕妇原发性感染与有症状的先天性疾病风险相关,且高发病率常与极低出生体重有关。无症状感染的新生儿在婴儿期会出现各种后遗症。这是阿根廷首次针对先天性和产后巨细胞病毒感染新生儿开展的研究。本研究的目的是评估聚合酶链反应(PCR)技术使用不同引物对检测组织培养中分离出的以及直接在尿液和干血斑(DBS)标本中分离出的巨细胞病毒的性能。将结果与IgM检测结果进行比较。
该研究于1999年至2001年在实验室对常规样本进行。从61名新生儿/婴儿中选取了61份尿液样本和56份血清样本,33名在出生后前两至三周内样本接受分析的患者被视为先天性感染;其余28名样本采集时间晚于第三周的患者被归为围产期感染,尽管仅在4例中明确确定了围产期感染传播。通过在人包皮成纤维细胞中从尿液样本中分离病毒来进行巨细胞病毒诊断。在病毒分离物中,使用针对IE、LA和gB基因的三种不同引物对进行人巨细胞病毒PCR检测。随后,使用gB引物对尿液和Guthrie卡片上的11份干血斑(DBS)样本直接进行PCR和巢式PCR(nPCR)检测,然后将这些结果与血清学结果进行比较。
33例先天性感染患者的主要临床表现为紫癜、黄疸、肝肿大和贫血。3例患者仅表现为低出生体重单一症状,10例有颅内钙化,2例有肾衰竭。在归为围产期感染的28例患者中,贫血、肝脾肿大和酶学改变较为突出,4例患者为HIV阳性。用于扩增gB区域的引物在巨细胞病毒分离物中的PCR阳性率为100%,而扩增IE和LA区域的引物的PCR阳性率分别为54%和61%。使用针对gB区域的引物对尿液样本(无需事先提取DNA)进行PCR扩增,检测到34/61份阳性样本。在33例先天性感染患者的样本中,24例(73%)呈阳性。当对这些样本使用nPCR时,全部呈阳性,而在其余28例患者中,发现2例阴性病例。还对11份样本的干血斑进行了巨细胞病毒DNA检测:7份DBS样本呈阳性,4份呈阴性。
针对gB片段区域的引物是检测阳性分离物中巨细胞病毒DNA的最佳选择。在先天性感染中,尿液直接PCR检测在高比例(73%)的样本中呈阳性;然而,在归为围产期感染的患者中,仅36%的病例呈阳性。使用n-PCR时,样本总阳性率达到97%。在干血斑中进行的PCR技术使4例患者的先天性感染得以确诊,其中3例得到证实。这些结果显示了nPCR在检测所有巨细胞病毒感染病例中的价值。该检测方法的优势在于可在正常工作日内进行,并能在比传统组织培养或空斑病毒检测所需时间短得多的时间内提供可靠结果。