Effectiveness of PCR primers for the detection of occult hepatitis B virus infection in Mexican patients

BackgroundOccult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HVB) DNA in the liver of HBsAg negative individuals with or without detectable viral DNA in serum. OBI is a diagnostic challenge as it is characterized by a very low viral load, intermittently detectable through time. Individuals with OBI can develop chronic hepatic disease, including liver cirrhosis and hepatocellular carcinoma. The aim of this work was to produce tools to improve OBI detection of the HVB genotypes prevalent in Mexico.MethodsWe designed and tested primers to detect OBI in serum samples by nested and real-time PCR. Conserved sites in the viral genome were determined by alignment of the most frequent HBV genotypes in Mexico (H, G/H, F and D) and primers spanning the entire viral genome were designed for first round and nested PCR. Primers were tested in serum samples of 45 patients not co-infected with hepatitis C virus or with HIV, out of a group of 116 HBsAg (-)/anti-HBc (+) individuals. Primers were also tested in a control group with chronic HBV. Nested PCR products obtained from HBsAg (-)/anti-HBc (+) were sequenced and used to design primers for real-time PCR (SYBR Green).ResultsThe most effective primer pairs to detect HBV products by nested PCR targeted ORF regions: PreS2/P, S/P, X/PreC, and C; while by real-time PCR they targeted ORF regions PreS2/P, S/P, X, and C. Out of the 45 HBsAg (-)/anti-HBc (+) patients tested, the viral genome was detected in 28 (62.2%) and 34 (75.5%), with nPCR and real-time PCR respectively.ConclusionPrimers designed for real-time PCR detected up to 75.5% of suspected OBI Mexican patients, with or without liver disease, which represents an improvement from previous PCR strategies.背景 隐匿性乙型肝炎感染（Occult hepatitis B infection, OBI）被定义为乙型肝炎病毒（hepatitis B virus, HBV）DNA存在于HBsAg阴性个体的肝脏中，伴或不伴血清中可检测到的病毒DNA。OBI是一项诊断难题，因其病毒载量极低且可间歇性检出。隐匿性乙型肝炎感染患者可进展为慢性肝脏疾病，包括肝硬化与肝细胞癌。本研究旨在开发相关工具，以提升墨西哥流行的HBV基因型相关隐匿性乙型肝炎感染的检测效能。 方法 我们设计并验证了可通过巢式PCR（nested PCR）与实时荧光定量PCR（real-time PCR）检测血清样本中隐匿性乙型肝炎感染的引物。通过比对墨西哥最常见的HBV基因型（H型、G/H混合型、F型与D型）确定病毒基因组的保守位点，并针对第一轮PCR与巢式PCR设计了覆盖整个病毒基因组的引物。我们从116名HBsAg(-)/抗-HBc(+)个体中选取45名未合并丙型肝炎病毒或人类免疫缺陷病毒感染的患者的血清样本对引物进行测试。同时，我们还在慢性HBV感染的对照组中对引物进行了验证。对从HBsAg(-)/抗-HBc(+)样本中获得的巢式PCR产物进行测序，并以此设计用于SYBR Green实时荧光定量PCR的引物。 结果 经巢式PCR检测HBV产物时，最有效的引物对靶向的开放读码框（open reading frame, ORF）区域为PreS2/P、S/P、X/PreC及C；而实时荧光定量PCR的引物则靶向PreS2/P、S/P、X及C区域。在45名HBsAg(-)/抗-HBc(+)受试患者中，巢式PCR与实时荧光定量PCR分别在28名（62.2%）与34名（75.5%）患者中检出了病毒基因组。 结论 针对实时荧光定量PCR设计的引物可在伴或不伴肝脏疾病的疑似隐匿性乙型肝炎感染墨西哥患者中实现最高75.5%的检出率，相较于此前的PCR检测策略实现了检测效能的提升。