Gashout Aisha, Amro Ahmad, Erhuma Mabruk, Al-Dwibe Hamida, Elmaihub Eanas, Babba Hamouda, Nattah Nabil, Abudher Abdalhafid
Faculty of Medical Technology Pathology Department, University of Tripoli, Tripoli, Libya.
Faculty of Pharmacy, Al-Quds University, Main Campus, Abu Dis, P.O. Box 5100, Jerusalem, Palestine.
BMC Infect Dis. 2016 Apr 16;16:157. doi: 10.1186/s12879-016-1491-5.
Toxoplasma gondii infections are prevalent in humans and animals throughout Libya. Current diagnosis is based on detection of Toxoplasma-specific IgM and IgG. In this study, we established and optimized a diagnostic PCR assay for molecular diagnosis of T. gondii in Libya.
From January to December, 2010, 177 blood and serum samples were collected from suspected patients. This includes: 140 women who have had spontaneous abortions, 26 HIV-positive patients, nine patients with leukemia and lymphoma, and two infants with ocular infection. Samples were screened for anti-Toxoplasma IgG and IgM antibodies before DNA extraction. The surface antigen gene 2 (SAG2) was targeted in a semi-nested PCR to amplify a 999 bp and a 614 bp fragment in the first and the second run respectively.
A total of 54/140 (38.5 %) women who have had spontaneous abortions, 23/26 (88 %) HIV patients, 6/9 (66.6 %) of the leukaemia and lymphoma patients, and one child with ocular infection were seropositive for anti-Toxoplasma IgG and/or IgM. Genomic DNA was extracted from 38 selected seropositive samples. The PCR was sensitive enough to detect DNA concentration of 12 ng/μL. PCR analysis was performed for 38 selected seropositive patients (16 women who have had spontaneous abortions, 15 positive HIV patients, six leukaemia patients and one child with ocular infection). Our designed primers were successfully amplified in 22/38 (57.9 %) samples; 5/12 (35.7 %) from serum and 17/26 (65.8 %) from whole blood samples. All PCR positive samples were IgG-positive except two samples which were IgM and IgG & IgM-positive serum samples respectively. The semi-nested PCR confirmed five more samples. These included two leukaemia and two HIV-positive whole blood samples and one serum sample from an aborted woman.
The ability of PCR to diagnose active toxoplasmosis is needed in immunocompromised patients and congenital toxoplasmosis cases, especially when serological techniques fail. For the first time in Libya, we established and optimized semi-nested PCR of SAG2 gene. The developed PCR method was able to detect as little as 12 ng/μL of T. gondii DNA and was useful to diagnose the diseases in women who have had spontaneous abortions, HIV-positive patients, patients with leukemia and lymphoma, and infants with ocular infection.
弓形虫感染在利比亚的人类和动物中普遍存在。目前的诊断基于检测弓形虫特异性IgM和IgG。在本研究中，我们建立并优化了一种诊断性PCR检测方法，用于利比亚弓形虫的分子诊断。
2010年1月至12月，从疑似患者中采集了177份血液和血清样本。这包括：140名自然流产的女性、26名HIV阳性患者、9名白血病和淋巴瘤患者以及2名眼部感染的婴儿。在提取DNA之前，对样本进行抗弓形虫IgG和IgM抗体筛查。表面抗原基因2（SAG2）是半巢式PCR的靶向基因，分别在第一轮和第二轮扩增出999bp和614bp的片段。
140名自然流产女性中有54名（38.5%）、26名HIV患者中有23名（88%）、9名白血病和淋巴瘤患者中有6名（66.6%）以及1名眼部感染儿童的抗弓形虫IgG和/或IgM血清学检测呈阳性。从38份选定的血清学阳性样本中提取了基因组DNA。PCR检测灵敏度足以检测到浓度为12ng/μL的DNA。对38名选定的血清学阳性患者（16名自然流产女性、15名HIV阳性患者、6名白血病患者和1名眼部感染儿童）进行了PCR分析。我们设计的引物在22/38（57.9%）的样本中成功扩增；血清样本中有5/12（35.7%），全血样本中有17/26（65.8%）。除了两份分别为IgM阳性和IgG及IgM阳性的血清样本外，所有PCR阳性样本均为IgG阳性。半巢式PCR又确认了5个样本。其中包括2份白血病全血样本、2份HIV阳性全血样本和1份流产女性的血清样本。
免疫功能低下患者和先天性弓形虫病病例需要PCR诊断活动性弓形虫病的能力，尤其是在血清学技术失败时。我们在利比亚首次建立并优化了SAG基因的半巢式PCR。所开发的PCR方法能够检测低至12ng/μL的弓形虫DNA，有助于诊断自然流产女性、HIV阳性患者、白血病和淋巴瘤患者以及眼部感染婴儿的疾病。
