生殖器疱疹患者HSV-2和anti-HSV-2检测的临床意义

生殖器疱疹患者HSV-2和anti-HSV-2检测的临床意义

【摘要】 　　目的 探讨生殖器疱疹（GH）患者HSV- 2 PCR及HSV-2抗体（包括IgG、IgM）检测的临床意义，评价其对GH诊断的重要性和实用性。方法 对182例生殖器部位有皮损的现症GH患者检测HSV- 2 PCR及HSV-2抗体（包括IgG、IgM）。结果 HSV- 2 PCR阳性检测率为90.6%（165/182），明显高于HSV- 2IgG阳性检测率68.7%（125/182）和HSV- 2IgM阳性检测率为14.3%（26/128）；初发患者HSV- 2 PCR阳性检测率为89.7%(61/68) 明显高于HSV- 2IgG阳性检测率39.7%(27/68)和HSV- 2IgM阳性检测率25%（17/68）；复发患者HSV- 2 PCR阳性检测率为95.6%（109/114）,HSV- 2IgG阳性检测率为100%（114/114）,两者几无差别,而HSV- 2IgM阳性检测率为0；23例皮损不明显患者HSV- 2IgG阳性检测率为78.3%（18/23），明显高于 HSV- 2 PCR阳性检测率26%（6/23）和HSV- 2IgM阳性检测率13%（3/23）。 结论 HSV- 2 PCR检测法对有典型临床症状如水疱和糜烂的GH阳性率高、诊断价值大，对临床症状不典型者阳性率低；HSV- 2IgG检测对无皮损和无症状的患者有较大的诊断价值；HSV- 2IgM对诊断GH感染的价值不大。

【关键词】 生殖器疱疹；HSV- 2 PCR；HSV- 2IgG；HSV- 2IgM

　　Clinical significance of detection of HSV-2 and anti-HSV-2 in genital herpes patients.

　　ZHANG Yan

　　(Hainan Provincial People's Hospital, Haikou 570102, Hainan, P. R. China)

　　Abstract：Objective To investigate the clinical significance of HSV-2 gene and antibody expression in genital herpes (GH) patients. Methods Polymerase chain reaction (PCR) and ELISA were used respectively to detect the expression of HSV-2 gene and the corresponding antibody in the 182 GH patients. Results The positive rate of HSV-2 gene expression, 90.6% (165/182) was much higher than that of anti-HSV-2 antibody, either 68.7% (125/182) for IgG type or 14.3% (26/182)for IgM type. Among the patients suffering GH for the first time, the positive rate of HSV-2 gene expression, 89.7% (61/68) was much higher than that of anti-HSV-2 antibody, either 39.7% (27/68) for IgG type or 25% (17/68) for IgM type. Whereas among the recurred patients, there was no significant difference in the positive rate between the expression of HSV-2 gene , 95.6%(109/114), and the corresponding antibody, 100%(114/114) for IgG type and 0 for IgM type. For the 23 patients with indefinite herpes, the positive rate of anti-HSV-2 IgG antibody, 78.3%(18/23), was significantly higher than that of HSV-2 gene, 26%(6/23), and anti-IgM antibody, 13%(3/23), as well. Conclusion Detection of HSV-2 gene expression might be of significance for the diagnosis of GH patients with characteristic clinical manifestations, whereas for the patients with indefinite manifestations, the IgG antibody might be of significance. In addition, IgM antibody against HSV-2 might be of little significance for the diagnosis of GH.

　　Key words：genital herpes; (GH)HSV-2;Polymerase chain reaction; (PCR)ELISA antibody IgG IgM

生殖器疱疹（GH）是由单纯疱疹病毒（HSV）感染引起的一种常见的性传播性疾病（STD），以生殖器周围出现疼痛性皮肤疱疹损害为主要临床表现。HSV-2是其主要病原体。由于感染后具有较高的复发率，已经成为感染率最高的性传播疾病之一。此病目前尚无满意的治疗方法，且可引起新生儿、胎儿的严重感染而日益受到关注。现在临床仍以检测HSV-2抗原和抗体作为主要辅助诊断指标。我科对182例GH患者的HSV-2抗原和抗体检测结果进行了分析和探讨。

　　1 资料和方法

　　1.1 病例来源

　　2005年5月～2006年12月我科门诊182例有新发水疱的现症GH患者，其中男110例，女72例，平均年龄34岁，最小年龄20岁，最大年龄62岁。初发患者68例，其中8例皮损不明显或结痂；复发患者114例，其中15例皮损不明显或结痂。发病时间1～9d，平均3d。

　　1.2 标本采集和方法

　　HSV-2 PCR检测用消毒棉拭子在生殖器皮损处取疱液或组织液，试剂采用中山大学达安基因股份有限公司生产的HSV- 2 PCR荧光法检测试剂盒，按说明进行操作。HSV-2抗体（包括IgG、IgM）检测抽取静脉血，分离血清待查，用德国医学实验诊断有限公司提供的ELISA试剂盒，按试剂盒说明操作。

　　1.3 统计学分析

　　采用χ2检验。

　　2 结果
　　
　　HSV- 2 PCR阳性检测率为90.6%（165/182）；HSV- 2IgG阳性检测率68.7%（125/182）；HSV- 2IgM阳性检测率为14.3%（26/128）。前者阳性检测率明显高于后两者，经统计分析有统计学差异（P＜0.01）。
　　
　　在初发患者中HSV- 2 PCR阳性检测率为89.7%(61/68); HSV- 2IgG阳性检测率39.7%(27/68)；HSV- 2IgM阳性检测率为25%（17/68）。前者阳性检测率明显高于后两者，经统计分析有统计学差异（P＜0.01）。
　　
　　在复发患者中：HSV- 2 PCR阳性检测率为95.6%（109/114）；HSV- 2IgG阳性检测率为100%（114/114）；HSV- 2IgM阳性检测率为0。HSV- 2 PCR阳性检测率与HSV- 2IgG阳性检测率比较无统计学差异（P＞0.05）。
　　
　　在23例皮损不明显或结痂的患者中：HSV- 2 PCR阳性检测率为26%（6/23）；HSV- 2IgG阳性检测率为78.3%（18/23）；HSV- 2IgM阳性检测率为13%（3/23）。HSV- 2IgG阳性检测率明显高于HSV- 2 PCR及HSV- 2IgM阳性检测率，经统计分析有统计学差异（P＜0.01）。

　　3 讨论

　　HSV- 2是生殖器疱疹的主要病原体，通过性接触传染。目前实验室诊断方法主要有HSV-2病毒细胞培养，抗原检测、分子生物学检测以及抗体检测,其中仍以病毒细胞培养为“金标准”［1］，可是由于其实验条件要求严格，成本高，且敏感性低等原因，还不能用于临床诊断。血清学检查是目前应用最多检查抗体的方法，PCR则常用于HSV- 2病原体的检测。本研究对HSV- 2两种检测方法进行分析比较显示：GH初发患者中HSV- 2 PCR法阳性率明显高于HSV- 2IgG和IgM法，可能因早期血清特异性抗体尚未形成有关；复发患者中HSV- 2 PCR法阳性率与HSV- 2IgG阳性率无明显差异，但HSV- 2IgM阳性率为0；有典型临床症状如水疱、糜烂者HSV- 2 PCR法阳性率明显高于HSV- 2IgG和IgM法；无典型临床症状如结痂者HSV- 2IgG法阳性率明显高于HSV- 2 PCR法阳性率。关于HSV- 2IgG和IgM：初次感染早期可产生HSV特异IgM抗体，一般持续6～8周，复发或再感染时，不出现IgM抗体［2］，因此IgM抗体的临床意义是说明HSV原发感染且病期较短，对复发患者无诊断意义；HSV- 2IgG在早期特异性抗体尚未形成时对诊断帮助不大，对复发患者HSV- 2IgG的检测阳性率几近100%，能检测无皮损和无症状的患者，是发现亚临床无症状HSV感染者的最可行手段［3～4］，对于防治HSV的性传播和母婴传播有着重要意义。
　　
　　总之，HSV- 2 PCR与血清HSV- 2IgG和IgM检测均是方便、快捷、有效的方法，但各有其自身的优缺点，需联合检测才能更好地发挥各自的优势。

【参考文献】
　　［1］叶顺章.性传播疾病的实验室诊断技术［M］.北京:科学技术出版社,2001，95.

　　［2］赵辩.临床皮肤病学［M］.南京:江苏科学技术出版社,2001，542.

　　［3］Morrow RA,Brown ZA. Common use of inaccurate antibody assays to identify infection status with herpes simplex virus type 2［J］. Am J Ohstet Gynecol，2005，193:361～362.

　　［4］Morrow RA,Friedrich D. Inaccuracy of certain commercial enzyme immunoassays in diagnosing genital infections with herpes simplex virus types 1 or 2［J］. Am J Clin Pathol，2003，120:839～844.

作者单位：海南省人民医院皮肤科，海南 海口 570102.
日期：2010年1月13日 - 来自[2008年第8卷第1期]栏目
High Seroprevalence of Herpes Simplex Virus Type 2 Infection in French Human Immunodeficiency Virus Type 1-Infected Outpatients

Unite de Virologie Medicale, Unite d'Hygiene Hospitaliere et Service des Maladies Infectieuses, Hpital Robert Debre, Centre Hospitalo-Universitaire de Reims, and IFR-53/EA-3798, Faculte de Medecine de Reims, Reims
Unite de Virologie Medicale et Service d'Immunologie Clinique, Hpital Europeen Georges Pompidou
Centre Medical de l'Institut Pasteur, Paris, France

ABSTRACT

Using commercially available herpes simplex virus (HSV) type-specific serological diagnostic tests, HSV type 2 (HSV-2) antibody prevalence was assessed in two parallel prospective studies including 534 human immunodeficiency virus type 1 (HIV-1)-infected outpatients living in two areas of northern France. In the first cohort of 434 subjects, 223 (51%) individuals demonstrated a positive HSV-2 serological status while 66 (66%) of 100 subjects in the second cohort were seropositive for HSV-2 (51 versus 66%; P = 0.08). Among the 223 HSV-2-seropositive subjects identified in the first study cohort, only 22 (10%) had suffered from recurrent anogenital lesions during the past 12 months while 154 (69%) had no clinical history of herpesvirus infection. Our findings demonstrate high proportions of subclinical and undiagnosed HSV-2 infection in HIV-1-infected individuals and suggest that HSV type-specific serological testing in the French HIV-1-infected subpopulation could be an efficient strategy to diagnose clinically asymptomatic HSV-2 infections.

TEXT

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted virus that is the most common cause of genital ulceration worldwide (9-11). Transmission is facilitated by the frequent recurrence of infectious episodes of subclinical viral shedding (9). Undiagnosed and untreated genital herpesvirus infection in pregnant women can lead to vertical transmission from mother to newborn, causing infant morbidity and mortality (10). Moreover, there is increasing evidence that HSV-2 infection could significantly enhance the rates of sexual transmission and acquisition of human immunodeficiency virus (HIV) in developing countries (11). The seroprevalence of HSV-2 antibodies varies considerably by population, and it has been shown that the prevalence of HSV-2 antibodies in both developed and developing countries has increased markedly over the past few years (5). In German HIV type 1 (HIV-1)-infected subpopulations, the seroprevalence of HSV-2 has been reported to be 47.9%, suggesting that genital secretions of European HIV-HSV-coinfected patients may be a common source of horizontal or vertical HSV transmission (13). Moreover, European HIV-1-HSV-2-coinfected individuals may constitute subpopulations at high risk for HIV transmission to HIV-negative exposed individuals (10). At the present time, the significance of HSV-2 infection in European HIV-1-infected subpopulations remains to be assessed. In this study, we evaluated for the first time HSV seroprevalence and risk factors for HSV-2 infection in French HIV-1-infected outpatients.

In the present study, 534 HIV-1-infected outpatients were prospectively enrolled in two hospital settings for routine follow-up. The first prospective study included a cohort of 434 consecutive outpatients (324 men [mean age, 38 years] and 110 women [mean age, 43 years]) attending the Reims University Medical Center and associated hospitals in the Champagne-Ardennes and Picardie provinces (northeastern France). The second prospective study included a cohort of 100 outpatients (63 men [mean age, 45 years] and 37 women [mean age, 39 years]) attending the Service d'Immunologie Clinique of the Hpital Europeen Georges Pompidou, Paris, France. Among the 534 study outpatients, 507 (95%) were European Caucasians and 27 (5%) were from Africa or Asia, where they had initially acquired HIV-1 infection. Signed informed consent was obtained from each study patient, and an institutional review board approved the two parallel clinical investigations. HSV-1 and HSV-2 type-specific serologic tests were carried out using two commercially available enzyme-linked immunosorbent assays (Champagne-Ardennes and Picardie provinces, SeroHSV-1 and SeroHSV-2 [BMD Diagnostics, Marne-la-Vallee, France]; Paris, HerpeSelect HSV-1 and HSV-2 [Focus Technologies, Eurobio, Courtaboeuf, France]) (1, 6).

Statistical analysis was performed using STATA version 7 Software (STATA Inc.). Comparison of quantitative variables was performed using Student's t test or the nonparametric Mann-Whitney U test when necessary. Chi-square or Fisher's exact tests were used for comparison of the discrete data, and the odds ratio (OR) and 95% confidence interval (CI) were also calculated. P values under or equal to 0.05 were considered significant. All the variables demonstrating a P value under or equal to 0.05 by univariate statistical analyses were then included in a forward stepwise logistic regression analysis, allowing the calculation of independent risk factors.

Among the 534 study subjects, the overall rates of HSV-1 and HSV-2 antibody prevalence were 86% and 59%, with 52% HSV-1-HSV-2 coinfection; HSV-1 and HSV-2 antibody seroprevalences were similar between males and females (92% versus 80% for HSV-1, P = 0.09; 62% versus 57% for HSV-2, P = 0.78, respectively). In the first cohort of 434 subjects, 223 (51%) individuals demonstrated a positive HSV-2 serological status while 66 (66%) of 100 subjects in the second cohort were seropositive for HSV-2 (51 versus 66%; P = 0.08). Among the 434 subjects from the Champagne-Ardenne and Picardie provinces, we conducted a case-control study focusing on demographic features and possible sexual risk factors for HSV-2 seropositivity (Table 1). In a univariate analysis, two variables were significantly associated with HSV-2 seropositivity, including an age above 45 years and high-risk sexual behavior. In a multivariate analysis, the variables age above 45 years and high-risk sexual behavior appeared as two independent risk factors for HSV-2 seropositivity (OR = 1.68, 95% CI = 1.13 to 2.49, P = 0.010, and OR = 1.93, 95% CI = 1.13 to 3.31, P = 0.016, respectively) (Table 1).

Among the 223 HSV-2-seropositive subjects identified in the first study cohort, only 22 (10%) had suffered from recurrent anogenital lesions during the past 12 months while 154 (69%) had no clinical history of herpesvirus infection and were totally unaware of their herpesvirus infection. Only 6 (3%) had suffered from another diagnosed sexually transmitted infection during the 12-month period before inclusion, whereas 12 (5%) of the 221 HSV-2-seronegative patients had suffered from clinically and biologically proven sexually transmitted diseases during the same period (3 versus 5%; P > 0.5). Among the 20 HSV-2-seropositive patients demonstrating genital or anal herpes lesions at the time of inclusion, herpes outbreaks appeared not to be associated with lack of antiretroviral therapy or with CD4 T-lymphocyte counts in peripheral blood (data not shown).

In the present serological survey, nearly two-thirds of the selected HIV-1-infected adults living in Paris and northeastern France were seropositive for HSV-2 infection. Similar rates of seroprevalence had been previously reported in American, African, and Asian HIV-1-infected subpopulations (5, 11). HSV-2 antibody prevalence has been reported to be 17% in the French general adult population (4) and to range from 4 to 24% in other, similar, European populations (5, 8). Our HSV-2 antibody prevalence appeared to be statistically significantly higher than that previously reported by Malkin et al. (4) in the French general population (17.2% of 12,735 subjects), even after the values were adjusted according to age or gender (P < 0.001). Taken together, our findings showed high rates of HSV-2 antibody prevalence in two cohorts of French HIV-1-infected outpatients, suggesting that HSV-2 infection may be markedly associated with HIV-1 infection in France. Two previous studies had reported that the seroprevalence rates of HSV-2 infection were 48% and 75% in U.S. and German HIV-1-infected subpopulations, respectively (3, 5). A further HSV seroepidemiological survey including a representative number of French cohorts of HIV-1-infected outpatients is needed to confirm our present findings. In the present study, only 22 (10%) of 223 HSV-2-HIV-1-coinfected outpatients from the first study cohort had a clinical history of genital herpes at the time of inclusion or within the 12-month period before. By contrast, the majority of HSV-2-seropositive patients (69%) were totally unaware of their genital infection at the time of inclusion whereas the remaining 47 patients (21%) were aware of their HSV-2 status by a history of past genital herpes outbreaks. These findings demonstrate an unexpectedly high proportion of subclinical and undiagnosed HSV-2 infection in HIV-1-infected individuals. In addition, the occurrence of HSV-2 outbreaks in the study population appeared not to be associated with lack of antiretroviral treatment or with circulating CD4 T-lymphocyte counts, suggesting that HSV-2 recurrences may be poorly or not influenced by antiretroviral treatment, as previously reported (7). Interestingly, multivariate statistical analysis revealed that HIV-1-infected subjects aged more than 45 years or with high-risk sexual behavior were more likely to be infected with HSV-2. These findings are consistent with results reported in several previous seroepidemiological studies which identified sexual behavior and age as risk factors for HSV-2 seropositivity (12). This likely reflects the association of risk of HSV-2 acquisition with the cumulative increase in the number of sexual partners and the duration of sexual activity in the context of the chronic nature of HSV-2 infection, particularly in association with unprotected sexual intercourse (9, 10). High rates of HSV-2 seroprevalence in the HIV-1-infected subpopulation could have major consequences for the risk levels of transmission and acquisition of HSV-2 or HIV-1 infection via sexual intercourse and consequently could have major implications for the medical care of HSV-2-HIV-1-coinfected patients. Indeed, genital herpes is now considered one of the major cofactors increasing the rate of HIV-1 transmission by the sexual route (12). Coinfected individuals appeared to be subject to high levels of asymptomatic HSV-2 genital infection, which could increase their genital infectivity for HIV-1 and therefore the rates of HIV transmission to potentially exposed HIV-1-negative sexual partners (10). Such a situation could be particularly critical in discordant heterosexual or homosexual couples in which one of the two partners is HIV-1-HSV-2 coinfected while the other is not infected with HIV-1 (2, 3, 10). In conclusion, the results of the present study demonstrate high proportions of subclinical and undiagnosed HSV-2 infection in French HIV-1-infected outpatients. Moreover, our findings suggest that HSV type-specific serological testing in the French HIV-1-infected subpopulation could be an efficient strategy to diagnose clinically asymptomatic HSV-2 infections and therefore to reduce the risk of HSV-2 and HIV-1 sexual transmission by convenient prophylactic counseling against unprotected intercourse. ACKNOWLEDGMENTS This study was independently conducted by the University Medical Center of Reims and was supported by GlaxoSmithKline Laboratory (Marly-le-roi, Paris, France) and by the Faculty of Medicine of Reims (IFR53/EA-3798) for clinical research. We acknowledge BMD diagnostics (Marne-la-Vallee, France) and Eurobio (Courtaboeuf, France) for providing HSV-2 type-specific enzyme-linked immunosorbent assay kits. REFERENCES Ashley, R. L. 2002. 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Virol. 61:201-207. 日期：2007年5月10日 - 来自[2005年第43卷第8期]栏目 Detection and Typing of Herpes Simplex Virus (HSV) in Mucocutaneous Samples by TaqMan PCR Targeting a gB Segment Homologous for HSV Types 1 and 2 Department of Clinical Virology, Gteborg University, Gteborg, Sweden ABSTRACT Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are major causes of mucocutaneous lesions and severe infections of the central nervous system. Here a new semiautomated method for detecting and typing of HSV was used to analyze 479 mucocutaneous swab samples. After DNA extraction using a Magnapure LC robot, a 118-bp segment of the gB region was amplified by real-time PCR utilizing type-specific TaqMan probes to identify HSV-1 or HSV-2. HSV detection in a single well using probes labeled with carboxyfluorescein (FAM) for HSV-1 and JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein) for HSV-2 had a sensitivity similar to that seen in separate reactions. All but one of 217 samples (99.5%) that had been positive by virus culture were positive by TaqMan PCR, with a correct identification of type in all cases. Out of 262 samples negative by virus culture, 48 (18.3%) were positive by TaqMan PCR, with higher Ct values compared with culture positive samples (P < 0.0001). Overall, the Ct values for HSV-1 were lower than for HSV-2 (mean, 25.5 versus 27.9), but to some extent this could be due to weaker fluorescence by JOE. Lower Ct values for HSV-1 were seen also in the 202 genital samples (79 HSV-1, 122 HSV-2, 1 HSV-1 and HSV-2), indicating that HSV-1 replicates as well as HSV-2 in the genital area. HSV-1 constituted 40% of genital infections and was associated with lower mean age (29.2 versus 36.4 years), probably reflecting the fact that recurrent genital HSV-1 infections are rare. INTRODUCTION Human herpes simplex virus (HSV) types 1 and 2 are common and important pathogens, which may cause severe disease in newborns and immunosuppressed patients. In immunocompetent subjects, both primary and reactivated infections are usually mild but may rarely spread to the central nervous system causing encephalitis, myelitis, or meningitis. HSV-1 typically causes orofacial blisters, keratitis, pneumonia, or encephalitis and has emerged as a common cause of genital herpes. HSV-2 typically causes genital lesions or meningitis (which may be recurrent). Both types may cause severe disease when transmitted perinatally. Detection of HSV DNA by PCR has become an important method for early diagnosis of infections in the central nervous system (10, 19), and has also been described as an alternative to viral culture for identifying HSV in mucocutaneous lesions (2, 6, 8). Typing can be done in the enzyme immunoassay format using type-specific antibodies, or by PCR techniques that specifically amplify either genotype in separate reactions (20) or distinguishes the amplicons by probes or melting point analysis (20). Recently, methods based on real-time PCR have been used for quantitating HSV (1, 11, 19), but the clinical value of this is not yet established. Here we report a new real-time PCR method based on amplification of a homologous segment of the gB region and distinction of HSV-1 and HSV-2 by the use of TaqMan probes. MATERIALS AND METHODS Samples. Cotton swabs were sent to the laboratory in buffered saline or viral transport medium. After viral culture inoculation the remaining volume was stored at –20°C until tested in this study. The majority (77%) of the samples were from genital lesions and were taken by gynecologists (the localization of the lesion was recorded for the 263 samples that turned out to be positive for HSV by PCR). Although sample handling and HSV diagnostics is well established in our city, a delay in sample transport and/or exposure to temperatures above 8°C could have occurred for a minority of the samples contributing to a negative virus culture. Virus culture. Two hundred microliters of sample (cotton swab in transport medium) were transferred to tubes with Green Monkey kidney cell lines (GMK-AH1). The cells were cultured in Eagle's minimal essential medium supplemented with 2% calf serum and antibiotics. The cells were examined for cytopathogenic effect daily for 7 days and positive samples were subtyped by using type-specific monoclonal antibodies against HSV-1-glycoprotein C and HSV-2 glycoprotein G (14). Extraction of HSV DNA. DNA was extracted in a Magnapure LC robot (Roche Diagnostics, Mannheim, Germany) using the Magnapure DNA Isolation Kit according to the manufacturer's instructions. The input and output volumes were set to 200 μl and 100 μl, respectively. In part of the study, freeze-thawing of the sample once was used as an alternative method for DNA preparation. In these cases 10 μl of the thawed sample was used in PCR without further procedures. Real-time PCR. A 118-nucleotide segment of the gB region was amplified by the use of primers described in Table 1. The distinction between HSV-1 and HSV-2 is based on differences between the probes at five nucleotide positions. The reaction volume of 50 μl contained 25 μl universal master mix (UMM, Applied Biosystems, Foster City, CA), 10 μl of sample DNA, and primers and probes at concentrations described in Table 1. Amplification was done in a real-time PCR instrument ABI Prism 7000 (Applied Biosystems). After incubation for 2 min at 50°C (uracil-N-glycosylase digestion) and 10 min denaturation at 95°C, 45 cycles of two-step amplification (15 s at 95°C, 60 s at 58°C) were performed. For each positive sample the Ct (threshold cycle) value was recorded. The Ct is the cycle when the fluorescence has become detectable and is in the exponential phase of amplification, and the Ct value is inversely proportional to the log concentration of target DNA. In the first part of this evaluation each sample was run in parallel reactions with FAM (6-carboxyfluorescein) labeled probes; one for HSV-1 using HSV1-F, HSV1&2-R and HSV1-probe and one for HSV-2 using HSV-2-F, HSV1&2-R, and HSV-2-probe. In the second part we used a reaction mixture that contained primers and probes for both HSV-1 and HSV-2, then using a HSV-2 probe labeled with JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein). In some experiments the samples were diluted prior to PCR to evaluate amplification efficiency or the impact of inhibitory substances. In additional experiments we evaluated the possibility to run the PCR (using the same reagents and setup) on a traditional PCR thermocycler (ABI 9700, Applied Biosystems), and use the real-time PCR instrument only for fluorescence reading, so-called post-PCR plate read. In this case, the PCR plate was transferred to the ABI Prism 7000 instrument directly after PCR, and fluorescence reading was carried out, using the "allelic discrimination" function in the SDS 7000 software. Sample testing. Three sets of samples were tested. First, 41 consecutive samples that were positive in viral culture (20 HSV-1 and 21 HSV-2) were analyzed. Each of these samples was tested in 16 positions: four duplicate reactions with the HSV-1 probe and the HSV-2 probe. These four reactions evaluated different extraction protocols: Magnapure extraction without or with a subsequent 1:10 dilution, and freeze-thawing without or with a subsequent 1:10 or 1:100 dilution. The second set consisted of 73 consecutive samples that were negative by viral culture. These samples were analyzed in duplicates for each of two sample preparations: Magnapure extraction (without subsequent dilution) or freeze-thawing with a subsequent 1:10 dilution. Finally, a third group was analyzed comprising 364 consecutive samples either positive (n = 205) or negative (n = 159) by virus culture. In this third group the reaction was run in a single well with a reaction mixture containing both a HSV-1 probe labeled with FAM and an HSV-2 probe labeled with JOE, and each sample was run without or with 1:4 predilution in water. RESULTS PCR efficiency and sensitivity. The overall sensitivity of a PCR test depends not only on the amplification efficiency of the PCR, but also on the capacity of the sample preparation procedure to extract DNA and remove inhibitory substances. In real-time PCR the amplification efficiency can be assessed by analyzing a given sample in serial dilution. Plotting the results should then yield a slope around 3.4, because in an optimal amplification, a tenfold dilution should correspond to 3.33 cycles. The amplification efficiency of the PCR was estimated by analyzing purified HSV DNA diluted in five 1:10 steps. The Ct values then ranged from 9.1 to 22.6 with an R2 of 0.998 and slope of 3.40 for HSV-1, and from 21.8 to 36.1 with an R2 of 0.996 and a slope of 3.55 for HSV-2, indicating good PCR efficiencies for both assays. To establish that the test had an acceptable sensitivity its performance was compared with the nested qualitative PCR that we use in clinical diagnostics (21). Two samples (representing HSV-1 or HSV-2) were diluted 1:2 in 5 steps and analyzed in duplicate by both the TaqMan assay and nested PCR. The end-point titer by the TaqMan assay was the same for HSV-1 and one 1:2 dilution step better than the nested PCR for HSV-2, indicating that the real-time PCR had a similar or better sensitivity than the nested PCR. Cross-reactivity and genotype mixtures. Ten culture-positive mucocutaneous samples (five HSV-1 and five HSV-2) were analyzed by PCR using a master mix for the nonmatching HSV type. Despite a very high virus concentrations in some samples (Ct range, 20 to 35 for HSV-1, 18 to 29 for HSV-2) no cross-reactions were seen. Cross-reactivity was also absent in the subsequent testing of clinical samples, even in the 20 samples with Ct values below 20. Mixtures representing different proportions of two samples (HSV-1 and HSV-2) which both had shown a Ct around 30 were tested. The minority strain could be detected at expected Ct values when diluted 1/9 or 1/81 (Table 2). Samples positive by viral culture tested in separate PCRs for HSV-1 and HSV-2. The samples were analyzed by TaqMan PCR after different pretreatments and dilutions. Firstly, we wanted to compare sample preparation by Magnapure with a simple method (freeze-thawing) we knew was used by others. The latter approach, which releases viral DNA by lysis, might have a reduced sensitivity due to inhibition or incomplete DNA yield. Secondly, we analyzed the samples with or without predilution as a means to estimate the amplification efficiency and degree of inhibition. Only one culture-positive sample (HSV-2) was negative by PCR. The remaining samples were positive after Magnapure extraction with mean Ct values of 22.5 ± 1.0 standard error of the mean; range, 14.6 to 35.1 (n = 20) for HSV-1 and 23.5 ± 0.7 (range, 18.1 to 29.2; n = 21) for HSV-2. After dilution 1:100 the mean Ct increased by 7.7 cycles indicating absence of inhibitors. Conversely, strong inhibition was observed in samples analyzed after only freeze-thawing. The majority (32/41) of these samples were negative by TaqMan PCR when analyzed without predilution, despite the very high viral loads documented by PCR after Magnapure extraction. However, after predilution 1:10 or 1:100 all the samples were reactive by PCR with mean Ct values around 30. The Ct values in freeze-thawed samples diluted 1:100 were only 1.5 cycles higher than in those extracted by Magnapure and diluted 1:100, indicating that most of the inhibition was lost at this dilution. In freeze-thawed samples diluted 1:10 inhibition was more pronounced and corresponded to 4.2 cycles (compared to extraction by Magnapure). Therefore, the overall sensitivity was only slightly lower in freeze-thawed samples diluted 1:100 compared to 1:10. Altogether, detection after Magnapure extraction without dilution before PCR had a considerably higher sensitivity than with any of the freeze-thawing strategies, with mean, Ct values being seven to eight cycles lower. Samples negative by viral culture tested in separate PCRs. In the initial testing of 73 culture-negative samples by TaqMan PCR for HSV-1 and HSV-2 in parallel reactions, 18 (24.7%) were positive by PCR after Magnapure extraction. The mean, Ct value was 31.6 for HSV-1 and 34.2 for HSV-2, i.e., overall significantly higher than for the 41 culture-positive samples described above (mean, 32.7 versus 23.0, P < 0.0001). TaqMan detection after freeze-thawing was positive in 8 samples, 4 HSV-1 (Ct 25 to 34), 4 HSV-2 (Ct 31 to 40). Thus, 10 samples were only positive by TaqMan PCR after Magnapure extraction. Typing in single well. Finally, detection of HSV-1 and HSV-2 in a single well was evaluated on a further consecutive 364 mucocutaneous samples, 189 (52%) of which were negative by virus culture. All the 175 samples that were positive by culture were also positive by TaqMan PCR; 73 HSV-1, 101 HSV-2, and 1 HSV-1/HSV-2 mixture. In the sample where TaqMan PCR showed coinfection the Ct was 24.1 for HSV-1 and 29.5 for HSV-2, and in this sample coinfection was identified also by virus culture. In addition, 30 of the 189 samples (15.9%) that were culture negative were positive by TaqMan PCR (17 HSV-1 and 13 HSV-2). The mean Ct values were 2.7 cycles lower for HSV-1 than for HSV-2 (mean, 25.5 ± 0.51 [± standard error of the mean] versus 28.2 ± 0.43). However, part of this difference was probably due to a weaker fluorescence by the JOE-marked HSV-2 probe (which increased the Ct by up to 1.8 cycles, not shown). As shown in Fig. 1, the Ct values were higher in culture-negative than in culture-positive samples (mean, 31.8 versus 24.0 for HSV-1, 33.9 versus 27.5 for HSV-2, P < 0.0001 for both). In parallel (i.e., in the same run) with detection in a single well with HSV-1-FAM/HSV-2-JOE probes, 15 of the culture-positive samples (5 HSV-1, 10 HSV-2) were analyzed also in separate wells with FAM-labeled probes for both HSV-1 and HSV-2 to compare sensitivity. Detection in a single well then produced slightly higher Ct values, with a Ct difference (one well versus separate reactions) of 0.94 ± 0.58 (± standard deviation) cycles for HSV-1, P = 0.022, and 0.54 ± 0.57 cycles for HSV-2, P = 0.015. This indicates that single-well detection has a marginally lower sensitivity (corresponding to less than one cycle) compared to detection in separate PCRs. We also tested the potential impact of inhibition by analyzing 246 of the samples with and without diluting the sample 1:4 in H2O after Magnapure extraction. The median Ct difference then was 2.0 (interquartile range, 1.6 to 2.4) indicating that inhibition in general was absent. However, inhibition might be present in a minor fraction, because 3 samples were positive only after predilution (Ct 34.0 to 38.7). On the other hand 3 other samples were positive only without predilution (Ct 35.0 to 41.2). Sample data and HSV type for all samples. Table 3 summarizes the TaqMan PCR results for all the 478 samples analyzed in this study. Overall, 18.3% (48/262) of culture-negative samples were positive by TaqMan PCR. Of the culture-negative samples, 81% of those from nearby clinics (<15 km) compared to 33% of those from distant clinics had a Ct value above 31. As shown in Table 4, the majority (76%) of the 264 that were PCR positive had been taken from genital lesions, and out of these 29.2% were HSV-1. As depicted in Fig. 2, the patients with HSV-1 were younger than those with HSV-2 (mean age, 36.4 versus 29.2 years), with the majority being between 15 and 25 years old. Evaluation of plate read procedure. The possibility to run the amplification on a traditional PCR instrument and do the post-PCR plate read on the ABI Prism 7000 instrument was evaluated on a set of 32 samples, which were also run in parallel as a real-time PCR. The mean, Ct was 29.3 ± 5.7 (± standard deviation) for the eight HSV-1 and 28.5 ± 4.0 for the seven HSV-2 samples (17 were PCR negative). The results from plate read after PCR on the 9700 instrument agreed well with plate read after real-time PCR on the 7000 instrument, (Fig. 3; Table 5), with similar fluorescence signals: The mean delta Rn ratio (ABI 7000/ABI 9700) was 1.17 ± 0.21 (± standard deviation) for HSV-1 and 1.08 ± 0.05 for HSV-2. However, one HSV-1 sample, which in real-time PCR had a Ct of 38.2 and was clearly identified as positive on plate read after PCR in the 7000 instrument (with a delta Rn of 3.67), showed a borderline reaction on plate read after PCR in the 9700 instrument (with a delta Rn of 2.34). DISCUSSION Here we present a real-time PCR method for detection and typing of herpes simplex virus in mucocutaneous lesions. The method comprises DNA extraction by a Magnapure LC robot, subsequent amplification of homologous segments of the gB genes of HSV-1 and HSV-2, and detection using TaqMan probes in a 96-well format on an ABI Prism 7000 real-time PCR instrument (Fig. 4). The primer regions in the targeted segment of gB are almost identical, but the probe region differs by 5 nucleotides between HSV-1 and HSV-2. This difference allowed genotyping without cross-reactivity even at very high virus concentrations (i.e., Ct values below 20, probably corresponding to virus concentrations above 108 copies/ml). An advantage of targeting homologous gene segments is that Ct values for of HSV-1 and HSV-2 are comparable and that mutual standards or control samples may be used. This would be of particular value if the assay is adapted to yield quantitative results, which by others has been done in separate reactions for HSV-1 and HSV-2 (19). In our laboratory, a nested PCR targeting the gD and gG regions has been used for the past 10 years for identifying HSV-1 and HSV-2 (21). In comparison with these tests, the real-time PCR had an equal or even slightly higher sensitivity. Probably, this high sensitivity is due to several factors, such as an efficient extraction protocol using Magnapure (7), a small amplicon size and Tm values for primers and probes within recommended limits. Compared with nested PCR the processing time is shorter, the contamination risk is less, and specificity is enhanced by hybridization. We also evaluated a very simple preparation of mucocutaneous samples, i.e., only freeze-thawing. We then noticed a marked inhibitory impact in mucocutaneous samples (5), which may originate either from substances in the sample or from the transport medium. Most of the inhibition disappeared when the sample was diluted 1:10 or 1:100 or when Magnapure extraction was used. The overall performance of freeze-thawing was acceptable and all but one of the culture-positive and some culture-negative samples were reactive by PCR after this preparation. Although we prefer the Magnapure extraction because it demonstrated an even higher sensitivity, the simplicity and lower cost make freeze-thawing preparation an attractive alternative, in particular if equipment for automated DNA extraction is lacking. In the later part of the study we used Magnapure extraction and coamplification in a single well with an HSV-1-probe labeled with FAM and an HSV-2-probe labeled with JOE. We then analyzed 364 consecutive clinical samples, comparing this method with virus culture and subsequent typing by enzyme immunoassay. TaqMan detection proved superior, because in addition to correctly identifying all samples that were positive by culture, 16% (30/189) of samples that were culture-negative were positive by PCR. Most of the culture-positive samples were reactive with low Ct values, indicating high virus concentrations. The mean Ct value was 2.5 cycles higher for HSV-2 than for HSV-1 indicating that virus secretion may be lower in HSV-2 than in HSV-1 lesions. However, part of this difference was calculated to be due to a weaker fluorescence from the JOE-marked HSV-2 probe. Still, the observation indicates that HSV-1 replicates similarily or at a slightly higher rate than HSV-2, also in the genital area from which the majority of the samples were collected. It should however be kept in mind that such a difference might be due to the fact that primary infections probably were more frequent among the HSV-1 positive samples. This could also be of relevance for the interpretation of the only case with dual (genital) infection in which the Ct value was 4.4 cycles lower for HSV-1 than for HSV-2. Our findings seem to contrast to data reported by van Doornum et al., who observed lower mean, Ct for HSV-2 than for HSV-1 (22), Therefore, further quantitation studies including samples from defined primary versus recurrent infection of the two subtypes are warranted. In accordance with previous reports from the Gteborg area (17), we found that 40% of the genital samples harbored HSV-1. Although clinical information about previous episodes were lacking, most of the HSV-1 cases were probably primary infections (15), while recurrences most likely constituted a substantial proportion of those with HSV-2 (16). This would fit well with the finding that patients with HSV-2 were older (P < 0.001) than those with HSV-1. This difference was however confined to women, suggesting that young women more frequently acquire primary genital HSV-1 infection by oral sex (13) (or possibly that recurrent HSV-2 is more frequent in older females). In accordance with previous findings (7, 22), the culture-positive samples in general had Ct values below 30, while a most of the culture-negative samples that were positive by TaqMan PCR had Ct above 30, indicating that a positive culture corresponds to a certain and relatively high concentration of virus. Because a Ct of 30 typically reflects a DNA concentration around 100,000 copies/ml, and 200 μl are used for virus culture, then in general more than 20,000 virions appears to be required for a positive culture. However, under favorable conditions one PFU of HSV corresponds to 10 to 100 virus particles (9), suggesting that only a small fraction (<1%) of the virions in the clinical samples were viable. In the cases where culture was negative despite Ct values below 30, the proportion of viable virions was probably even lower due to unfavorable transport conditions. This is supported by the fact that culture-negative samples with low Ct values were more often from more distant clinics. Real-time PCR of HSV has previously been used by others. Ryncarz et al. (19) used TaqMan probes, targeting a conserved part of gB for quantification and a segment of gG for typing. Others have used TaqMan probes to identify different amplicons for HSV-1 and HSV-2. For example, Weidman et al. targeted gD1 and gG2 (23), while van Doornum et al. targeted gG1 and gD2 (22). Our assay amplifies homologous regions with few sequence differences between HSV-1 and HSV-2 apart from five positions in the probe region, allowing quantification and typing in one reaction. This strategy may also be applied on a LightCycler instrument, where the type can be identified by melting point analysis using SYBR green (2, 18) or hybridizing FRET probes (6, 8). Of these methods, the hybridizing probes have the advantage of a higher specificity, but the relatively large probing segment increases the risk of mismatches and unexpected melting temperatures (3). At present, the LightCycler instrument also has a limited processing capacity, as only 32 samples can be analyzed in each run. In addition to the larger sample size itself, an advantage with the 96-well format is that the PCR may be run on a traditional thermocycler, followed by fluorescence detection using post-PCR plate read. This option should be of value for laboratories that have a limited real-time PCR capacity, as it reduces the demand for time on the instrument to around 10 min. In summary, detection and typing of HSV by this new TaqMan PCR after Magnapure extraction of DNA had a sensitivity superior to virus culture and equivalent to that of a nested qualitative PCR. Detection of HSV-1 and HSV-2 in a single well had a sensitivity similar to detection in separate wells, an approach which is preferable because reagent costs are reduced and through-put may be increased to 96 samples in each run. The high sensitivity in combination with high specificity and capacity make it suitable for clinical diagnosis of HSV in mucocutaneous lesions. ACKNOWLEDGMENTS We thank Maria Andersson for technical expertise. REFERENCES Aberle, S. W., and E. Puchhammer-Stockl. 2002. Diagnosis of herpesvirus infections of the central nervous system. J. Clin. Virol. 25 Suppl. 1:S79-85. Aldea, C., C. P. Alvarez, L. Folgueira, R. 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Microbiol. 41:1565-1568. 日期：2007年5月10日 - 来自[2005年第43卷第5期]栏目 Rapid Diagnosis of Herpes Simplex Virus Infection by a Loop-Mediated Isothermal Amplification Method Department of Pediatrics, Fujita Health University School of Medicine Department of Medical Information Technology, Fujita Health University College, Toyoake Department of Dermatology, Central Hospital of Tokai Medical Institute, Tokai Department of Virology, Nagoya University Graduate School of Medicine, Nagoya, Aichi Department of Obstetrics and Gynecology, University Hospital, Mizonokuchi, Teikyo University School of Medicine, Kawasaki, Kanagawa, Japan ABSTRACT Primers for herpes simplex virus type 1 (HSV 1)-specific loop-mediated isothermal amplification (LAMP) method amplified HSV-1 DNA, while HSV-2-specific primers amplified only HSV-2 DNA; no LAMP products were produced by reactions performed with other viral DNAs. The sensitivities of the HSV-1- and HSV-2-specific LAMP methods, determined by agarose gel electrophoresis, reached 500 and 1,000 copies/tube, respectively. The turbidity assay, however, determined the sensitivity of the HSV-1- and HSV-2-specific LAMP methods to be 1,000 and 10,000 copies/tube, respectively. After initial validation studies, 18 swab samples (in sterilized water) collected from patients with either gingivostomatitis or vesicular skin eruptions were examined. HSV-1 LAMP products were detected by agarose gel electrophoresis in the 10 samples that also demonstrated viral DNA detection by real-time PCR. Nine of these 10 samples exhibited HSV-1 LAMP products by turbidity assay. Furthermore, both the agarose gel electrophoresis and the turbidity assay directly detected HSV-1 LAMP products in 9 of the 10 swab samples collected in sterilized water. Next, we examined the reliability of HSV type-specific LAMP for the detection of viral DNA in clinical specimens (culture medium) collected from genital lesions. HSV-2 was isolated from all of the samples and visualized by either agarose gel electrophoresis or turbidity assay. TEXT Viral isolation and serological assays are standard methods of herpes simplex virus (HSV) diagnosis. Both viral isolation and serological testing, however, require substantial time to obtain accurate final results. More rapid detection has been achieved by modification of cell culture techniques by centrifugation of inocula on cell monolayers and the use of immunofluorescence techniques (6). Recent studies have suggested that detection of HSV DNA by PCR increases the sensitivity of viral infection detection compared to antigenic detection or cell culture methods (3, 4, 11, 13, 14). While quantitative analysis of viral DNA by real-time PCR may become a valuable tool for bedside monitoring of HSV infection and progression (1, 2, 7, 10, 17, 21, 22), it has not yet become a common procedure in hospital laboratories due to the requirement of specific expensive equipment (a thermal cycler). Recently, Notomi et al. (18) reported a novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which is used to amplify DNA under isothermal conditions with high specificity, efficiency, and speed. The most significant advantage of LAMP is the ability to amplify specific sequences of DNA between 63 and 65°C without thermocycling. Thus, the technique requires only simple and cost-effective equipment amenable to use in hospital laboratories. The LAMP method also exhibits both high specificity and high amplification efficiency. As the LAMP method uses four primers which recognize six distinct target DNA sequences, the specificity is extremely high. This method also exhibits extremely high amplification efficiency, due in part to its isothermal nature; as there is no time lost due to changes in temperature and the reaction can be conducted at the optimal temperature for enzyme function, the inhibition reactions that often occur at later stages of typical PCR amplifications are less likely to occur. Thus, this method could potentially be a valuable tool for the rapid diagnosis of infectious diseases (5, 8, 9, 12, 19, 23) in both commercial and hospital laboratories. In this study, we sought to establish a LAMP-based HSV type-specific DNA amplification method and examine its reliability for the detection of HSV DNA from clinical specimens. HSV-1 (KOS) DNA and HSV-2 (186) DNA were used as positive controls to determine the appropriate conditions for HSV type-specific LAMP and to establish the baseline sensitivity and specificity levels. HSV-1 (KOS), HSV-2 (186), varicella-zoster virus (VZV) (Oka), human cytomegalovirus (HCMV) (AD-169), human herpesvirus type 6B (HHV-6B) (Z29), and HHV-7 (RK) DNA were used to determine the specificity of HSV type-specific LAMP. Plasmids containing the HSV-1 and HSV-2 target sequences were used to determine the assay sensitivity. To determine the reliability of HSV type-specific LAMP for detection of viral DNA from clinical samples, 18 swab samples (sample numbers 1 to 18) were collected from patients with either gingivostomatitis or vesicular skin eruptions. Swabs were collected from patients at the outpatient clinic of the Fujita Health University hospital and the Central Hospital of the Tokai Medical Institute and placed into 1 ml of sterilized water. Five swab samples (sample numbers 19 to 23) were also collected from patients with genital herpes at Teikyo University Mizonokuchi Hospital outpatient clinic. Swabs were collected from the lesions and placed into culture medium. HSV-2 was isolated from all of these samples. We attempted detection of HSV-1 and HSV-2 DNA from either post-DNA extraction or without DNA extraction by using HSV-1-specific and HSV-2-specific LAMP. The results of HSV type-specific LAMP were compared with results obtained by the previously established technique of HSV type-specific real-time PCR to assess the reliability of the methods for the rapid diagnosis of HSV infection. LAMP reactions were conducted as described previously by Notomi et al. (18) and Nagamine et al. (16). The LAMP method requires a set of four primers (B3, F3, BIP, and FIP) that recognize a total of six distinct sequences (B1 to B3 and F1 to F3) within the target DNA. Primers for HSV-1 and HSV-2 LAMP were designed against the HSV-1 glycoprotein G (gG) and HSV-2 gG genes, respectively, by using Primer Explorer V software (FUJITSU, Tokyo, Japan), the locations and sequences of which are shown in Table 1. Primer BIP for the gG genes of HSV-1 (HS1BIP) and HSV-2 (HS2BIP) contained the B1 direct sequence and B2 complementary sequence, each specific for the respective strains. Primer FIP for the gG genes of HSV-1 (HS1FIP) and HSV-2 (HS2FIP) contained the F1 complementary sequence and the F2 direct sequence. Primers B3 (HS1B3 and HS2B3) and F3 (HS1F3 and HS2F3) for the HSV-1 and HSV-2 gG genes were located outside the F2-B2 regions. As additional loop primers increase the amplification efficiency (16), loop primers specific for the HSV-1 gG (HS1LPB and HS1LPF) and HSV-2 gG (HS2LPB and HS2LPF) genes were also synthesized. HS1LPB and HS2LPB contained the LPB sequence, while HS1LPF and HS2LPF contained the LPF complementary sequence. The LAMP reaction was performed by using a Loopamp DNA amplification kit (Eiken Chemical, Tochigi, Japan). Reaction mixtures (25 μl) contained 1.6 μM each FIP and BIP primer, 0.8 μM each outer primer (F3 primer and B3 primer), 0.8 μM each loop primer (LPF primer and LPB primer), 2x reaction mix (12.5 μl), Bst DNA polymerase (1 μl), and 5 μl of each sample. The mixture was incubated at 63°C for 30 min. Next, a TERAMECS LA200 (Teramecs, Kyoto, Japan) was used to measure turbidity after 30 min of LAMP (15). After turbidity measurement, LAMP products were subjected to electrophoresis on 1.5% agarose gels. Gels were visualized under UV light after ethidium bromide staining. To avoid contamination between samples, different rooms were used for DNA extraction, LAMP setup, and gel analysis using filter-containing pipette tips for aerosol protection. As the turbidities of five negative samples were demonstrated to be 0.01 ± 0.02, we defined 0.1 as the cutoff value for discrimination between positive and negative samples. Real-time PCR quantitated the amount of either HSV-1 or HSV-2 DNA in each sample. The genes encoding HSV-1 and HSV-2 glycoprotein G were selected for HSV type-specific real-time PCR. The sequences of the primers and probes used for these experiments were described previously by Pevenstein et al. (20). To develop an effective assay for rapid measurement of HSV DNA content, we first evaluated the specificity of our HSV type-specific primers. HSV type-specific LAMP was performed on DNA extracted from HSV-1 (KOS)-, HSV-2 (186)-, VZV (Oka)-, HCMV (AD-169)-, HHV-6B (Z29)-, and HHV-7 (RK)-infected cells. As the LAMP products contained several inverted-repeat structures, positive samples exhibit multiple bands of different sizes upon agarose gel electrophoresis. HSV-1-specific primers amplified only HSV-1 DNA and HSV-2-specific primers amplified only HSV-2 DNA (Fig. 1), and no LAMP products were detected in reactions performed with DNA from other viral infections. We also tested the specificity of the primers by using a turbidity assay. The use of HSV-1-specific primers elevated sample turbidity only in HSV-1 DNA-containing samples. Similar specificity was observed for HSV-2-specific primers (Fig. 1). We also determined the sensitivity of this method. Serial dilutions of either pGEMHS1 or pGEMHS2 plasmid containing the target sequences determined the detection limits of HSV type-specific LAMP. The sensitivities of the HSV-1- and HSV-2-specific LAMP determined by agarose gel electrophoresis were 500 and 1,000 copies/tube, respectively (Fig. 2). Detection by the turbidity assay, however, produced sensitivity levels of 1,000 and 10,000 copies/tube for HSV-1- and HSV-2-specific LAMP, respectively. After these initial validation studies, we determined the reliability of this HSV type-specific LAMP as a method of viral DNA detection from clinical specimens. Eighteen swab samples (sample numbers 1 to 18) collected from patients with either gingivostomatitis or vesicular skin eruptions were examined (Table 2). Neither HSV-1 nor HSV-2 LAMP products were detected in samples (sample numbers 1 to 8) from which no HSV DNA could be detected by real-time PCR. In contrast, HSV-1 LAMP products were detected by agarose gel electrophoresis in the 10 HSV-1-positive samples, correlating perfectly with the results of real-time PCR. When the turbidity assay was used, HSV-1 LAMP products were detected in all but 1 (sample number 10) of these 10 positive samples. No HSV-2 DNA could be detected in these samples by either real-time PCR or LAMP. As rapidity and simplicity of the method are critical for commercial and hospital laboratory use, we investigated the requirement for DNA extraction in HSV type-specific LAMP. Either agarose gel electrophoresis or turbidity assay directly detected HSV-1 LAMP product in all 10 swab samples (sample numbers 9 to 18) (sterilized water), with the exception of sample number 10, regardless of the presence or absence of DNA extraction (Table 2). As genital herpes is another important clinical manifestation of herpes infection, we next examined the reliability of HSV type-specific LAMP for the detection of viral DNA in clinical specimens (culture medium) collected from genital lesions (sample numbers 19 to 23) (Table 2). High copy numbers of HSV-2 DNA (ranging between 641 and 443,963 copies/tube) were detected in these samples by HSV-2 type-specific real-time PCR. Both agarose gel electrophoresis and turbidity assay detected HSV-2 LAMP products in all of the samples. To determine the necessity of DNA extraction by this method, we again tried to detect HSV-2 LAMP products in the samples with or without (culture medium) DNA extraction. In contrast, while HSV-2 LAMP products were detected in samples after DNA extraction, no HSV-2 LAMP products were detected in the samples without DNA extraction (Table 2). To determine if the culture medium contained an inhibitor of LAMP, we attempted to detect HSV LAMP products from both sterilized water and culture medium containing plasmid DNA which contained the target sequences. Although both HSV-1 and HSV-2 LAMP products could be detected in sterilized water containing the target sequences, no LAMP products were detected in culture medium containing these DNAs (data not shown). HSV-1- and HSV-2-specific LAMP specifically amplified HSV-1 and HSV-2 DNA, respectively, exhibiting no cross-reactivity with other human herpesviruses, including another member of the subfamily Alphaherpesvirinae, VZV (Fig. 1). This specificity was confirmed by agarose gel electrophoresis and turbidity assay. Although the capability to distinguish between HSV-1 and HSV-2 infection is not crucial for correct administration of antiviral drugs, this discrimination is important from an epidemiological or public health standpoint. As a consequence of the experiment used for determination of the assay sensitivity, it was suggested that the turbidity assay is a less sensitive detection method than agarose gel electrophoresis, as previously suggested (23). However, the turbidity assay is more appropriate for bedside monitoring due to its ease and rapidity. Additionally, turbidity measurement of LAMP products allows a reduction in operation time and reduces contamination risks because of the absence of agarose gel electrophoresis. We also evaluated the reliability of HSV type-specific LAMP in the detection of viral DNA from different clinical specimens. Although HSV-1 LAMP products were detected by agarose gel electrophoresis in 10 of the 18 swab samples (sterilized water) collected from patients with vesicular skin lesions and gingivostomatitis suspected as HSV infection, no HSV-2 LAMP products were detected in these samples. All five swab samples (culture medium) collected from the lesions of patients with genital herpes contained HSV-2 LAMP products. These results corresponded well with those from real-time PCR analysis, suggesting that HSV type-specific LAMP is a reliable method for the detection of viral DNA in clinical samples. Although an HSV-1 LAMP product could not be detected in one HSV-1-positive sample by turbidity assay, this inconsistency is probably due to low copy numbers. As the majority of clinical samples (e.g., skin eruptions, oral ulcers, and genital lesions) contain large quantities of viral DNA, the sensitivity of type-specific LAMP by turbidity assay is likely sufficient for the evaluation of most clinical samples. Moreover, all amplification steps are completed within 30 min with an LA-200, and it is a cheaper piece of equipment than that required for real-time PCR, which are major advantages for hospital laboratory use. Interestingly, when the swabs were collected in sterilized water, HSV LAMP products could be detected directly from the samples without DNA extraction. In contrast, LAMP products could not be detected directly from the culture medium containing viral DNA, regardless of the HSV strain. DNA target sequences, however, became detectable in samples after DNA extraction, suggesting that culture medium contains inhibitors of the LAMP reaction. As the DNA extraction step requires approximately 30 min, omission of DNA extraction could save both time and labor for preparing the samples for LAMP, a major advantage for rapid diagnosis in hospital laboratories. To our knowledge, this is the first report to demonstrate direct amplification of viral DNA from sterilized water containing viral nucleic acids without DNA extraction. We emphasize that the swab should be placed into sterilized water for direct amplification of viral DNA by LAMP. Direct amplification from swab samples in combination with assessment by turbidity assay would accomplish the entire amplification within 30 min. This system would therefore allow large increases in throughput, which is highly relevant for clinical laboratory use. Furthermore, as HSV DNA could be directly detected from swab samples without DNA extraction, the lesions of HSV infection may contain a large quantity of naked viral DNA as well as complete virions. Thus, direct detection of viral DNA from swab samples may be possible by additional DNA amplification methods such as real-time PCR. Further investigation will be necessary to confirm this hypothesis in the future. ACKNOWLEDGMENTS We thank Eiken Chemical for their contributions to this work. We also thank Akiko Yoshikawa and Maki Sawamura for their technical assistance. This work was supported in part by a grant-in-aid for the 21st Century COE Program of Medicine of Fujita Health University and the Open Research Center of Fujita Health University from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and also by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. REFERENCES Aldea, C., C. P. Alvarez, L. Folgueira, R. Delgado, and J. R. Otero. 2002. Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal. J. Clin. Microbiol. 40:1060-1062. 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Suga, Y. Enomoto, R. Suzuki, Y. Nishiyama, and Y. Asano. 2004. Detection of human herpesvirus 7 DNA by loop-mediated isothermal amplification. J. Clin. Microbiol. 42:1348-1352. 日期：2007年5月10日 - 来自[2005年第43卷第2期]栏目 Secreted Portion of Glycoprotein G of Herpes Simplex Virus Type 2 Is a Novel Antigen for Type-Discriminating Serology Department of Virology, Göteborg University, S-413 46 Göteborg, Sweden Received 12 March 2003/ Returned for modification 26 April 2003/ Accepted 11 May 2003 　 ABSTRACT Top Abstract Introduction Materials and Methods Results Discussion References The secreted portion of glycoprotein G (sgG-2) of herpes simplex virus type 2 (HSV-2) was evaluated as a novel antigen in an enzyme-linked immunosorbent assay (ELISA) format for detection of type-specific immunoglobulin G (IgG) antibodies in HSV-2-infected patients. The results were compared with those obtained by a commercially available assay, the HerpeSelect 2 ELISA (the FOCUS2 assay). Five different panels of sera were analyzed: panel A consisted of 109 serum samples from patients with a culture-proven HSV-1 infection that were Western blotting (WB) negative for HSV-2; panel B consisted of 106 serum samples from patients with a culture-proven recurrent HSV-2 infection that were WB positive for HSV-2; panel C consisted of 100 serum samples with no detectable IgG antibodies against HSV-1 and HSV-2; panel D consisted of 70 HSV-2 negative "tricky" serum samples containing antinuclear IgG antibodies or IgM antibodies against other viruses or bacteria; and panel E consisted of consecutive serum samples from 21 patients presenting with a first episode of HSV-2-induced lesions. When sera in panels A to C were analyzed, the sgG-2 ELISA and the FOCUS2 assay both showed sensitivities and specificities of 98%. In total, among the samples in panel D, 13 serum samples (19%) were false positive by the FOCUS2 assay and 1 serum sample (1.4%) was false positive by the sgG-2 ELISA. When the sera in panel E were analyzed, the sgG-2 ELISA detected seroconversion somewhat later than WB or the FOCUS2 assay did. We conclude that sgG-2 induces an HSV-2 type-specific antibody response and can be used for type-discriminating serology. 　 INTRODUCTION Top Abstract Introduction Materials and Methods Results Discussion References Herpes simplex virus (HSV) type 2 (HSV-2) infection is one of the most common sexually transmitted diseases in the world (3, 24). In the United States the prevalence of HSV-2 infection increased 30% between 1976 and 1994, and HSV-2 infection is detected in one of five persons aged 12 years or older (10). The same trend was reported among women in Sweden, where the HSV-2 antibody prevalence increased from 19 to 33% between 1969 and 1989 (11). In some African countries, the prevalence of HSV-2 has been reported to vary between 17 and nearly 70% (13). As HSV-2-induced lesions facilitate the transmission of human immunodeficiency virus (6, 32), HSV-2 infection poses an additional threat to these populations. One obstacle to the prevention of the transmission and spread of HSV-2 is that in the majority of cases infection is transmitted without the appearance of symptoms in the newly infected host (27, 33). In this situation, detection of HSV-2 type-specific antibodies may be the only available method for establishment of a correct diagnosis. Type-specific serological assays are also essential for estimation of the seroprevalence in epidemiological studies, for counseling of patients attending a sexually transmitted disease clinic, and for HSV-2 vaccine follow-up programs. Furthermore, a type-specific assay is warranted to discriminate between primary or recurrent HSV-2 infection. This is of special importance for pregnant women, as ongoing primary HSV-2 infection during delivery represents a considerable threat to the newborn (7, 8). Several of the viral envelope proteins of HSV-2 have been shown to be immunogenic, eliciting an antibody response in humans (2). Due to a high degree of genetic similarity between HSV-1 and HSV-2, most viral proteins induce a cross-reactive antibody response. Glycoprotein G of HSV-1 (gG-1) and HSV-2 (gG-2) is the only known viral envelope protein which elicits a type-specific antibody response. In the virus-infected cell, gG-2 is cleaved into a secreted amino-terminal portion (sgG-2) and a carboxy-terminal portion. The latter protein is further O-glycosylated, generating the cell membrane-associated mature gG-2 (mgG-2) (5, 28). The mgG-2 protein has widely been used as a prototype antigen for detection of type-specific antibodies against HSV-2 (2, 4, 12, 17). We recently showed that monoclonal antibodies (MAbs) directed against the sgG-2 protein identified type-specific linear and nonlinear epitopes that were devoid of cross-reactivity to HSV-1 antigens. In addition, a type-specific immunoglobulin G (IgG) antibody response was detected in sera from HSV-infected patients by using sgG-2 as the antigen (18). In the present study, the performance of immunosorbent purified sgG-2 in an enzyme-linked immunosorbent assay (ELISA) format (sgG-2 ELISA) was evaluated with large panels of sera collected from patients with isolation-proven HSV-1 or HSV-2 infections. These serum panels were also analyzed by the commercially available HerpeSelect 2 ELISA (the FOCUS2 assay; Focus Technologies, Cypress, Calif.), which is approved for use by the U.S. Food and Drug Administration. In addition, consecutive serum samples derived from patients presenting with a first episode of HSV-2-induced lesions were analyzed by different gG-2 based assays. 　 MATERIALS AND METHODS Top Abstract Introduction Materials and Methods Results Discussion References Cells and viruses. African green monkey kidney (GMK-AH1) cells and human epidermoid (HEp-2) cells were cultured in Eagle's minimal essential medium supplemented with 2% calf serum and antibiotics. A local wild-type HSV-2 isolate B4327UR was used (16). Serum samples. Sera were collected from clinical specimens received at the Department of Clinical Virology (Sahlgrenska University Hospital, Göteborg, Sweden). Five panels of sera were analyzed. Panel A consisted of 109 serum samples from patients with a culture-proven HSV-1 infection and with no detectable antibodies against HSV-2 by the Western blotting (WB) technique. Panel B consisted of 106 serum samples from patients with recurrent genital culture-proven HSV-2 infection that were WB positive for HSV-2. Panel C consisted of 100 serum samples which were HSV-1 and HSV-2 seronegative by ELISA with an HSV-1-derived type-common sodium deoxycholate-solubilized membrane preparation (15) and Helix pomatia lectin-purified mgG-2 (HPLmgG-2) as antigens. Panel D consisted of 70 "tricky" serum samples, with 19 serum samples containing IgM antibodies against Mycoplasma pneumoniae, 20 serum samples containing IgM antibodies against Epstein-Barr virus (EBV), 20 serum samples containing IgM antibodies against cytomegalovirus (CMV), and 11 serum samples containing antinuclear IgG antibodies (ANA). All serum samples were HSV-2 negative by WB and the HPLmgG-2 ELISA. IgM antibodies were detected by in-house assays by the immunofluorescence technique. Briefly, monolayers of P3HR1 cells infected with EBV and human embryonic cells infected with CMV were permeabilized and fixed in acetone. Serum samples were diluted in phosphate-buffered saline and incubated for 1 h at 37°C. Fluorescein isothiocyanate-labeled goat anti-human IgM (Jackson ImmunoResearch Laboratories) was used as the conjugate. Glycolipid antigens from M. pneumoniae were extracted with chloroform, methanol, and water and used for detection of IgM antibodies in an ELISA format. Alkaline phosphatase-conjugated anti-human IgM (DAKO) was used as the conjugate, and p-nitrophenyl phosphate disodium was used as the substrate. The sera were initially preadsorbed with GullSORB (Meridian Diagnostics Inc.). ANA were detected at the Department of Clinical Immunology, Göteborg, Sweden, by an indirect immunofluorescence technique with HEp-2 cells. Fluorescein isothiocyanate-labeled goat anti-human IgG (Jackson ImmunoResearch Laboratories) was used as the conjugate. Panel E consisted of serum samples from 21 patients who presented with a culture-proven first episode of HSV-2-induced lesions and whose acute-phase sera had no detectable antibodies, as determined by the HPLmgG-2 ELISA. In total, 52 consecutive serum samples were collected. The acute-phase blood samples were drawn close to the time (±7 days) or at the time (day 0) that the sample used for virus isolation was obtained. The number of serum samples drawn from each patient varied between two and five, and the time from the drawing of the first sample to the time of drawing of the last sample ranged from 2 to 545 days. Typing of clinical HSV-1 and HSV-2 isolates. The isolates were cultured on GMK-AH1 cells and typed as described previously by using anti-HSV-1 and anti-HSV-2 MAbs (20). WB. The WB technique is considered the "gold standard" for the detection of HSV-2-specific IgG antibodies (2). Antigens were prepared for WB by infecting HEp-2 cells with HSV-2 isolate B4327UR, as described previously (19). Briefly, the antigens were mixed with sample buffer containing sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis under reducing conditions by using NuPAGE 7% Tris-acetate gels (Novex). The proteins were electrotransferred to an Immobilon-P transfer membrane (Millipore Corp.). The strips were incubated overnight with sera at a 1:100 dilution. A serum sample drawn from an individual with culture-confirmed HSV-2 infection and an anti-mgG-2 MAb (21) were used for correct identification of the carboxy-terminal intermediate portion of gG-2 and the mgG-2 protein. Peroxidase-labeled rabbit anti-human or rabbit anti-mouse IgG (DAKO) was used as the conjugate, and 4-chloro-1-naphthol was used as the substrate. A positive WB profile was defined as reactivity to mgG-2 (120 kDa) alone or in combination with reactivity to the carboxy-terminal intermediate portion of gG-2 (70 kDa) (21). HPLmgG-2 ELISA. HPLmgG-2 was coated on Maxisorp microtiter plates, and the assay was performed as described previously (21, 29). sgG-2 ELISA. Immunosorbent affinity chromatography-purified sgG-2 (0.75 mg/ml) was prepared as described earlier (18) and coated at a 1:3,000 dilution in carbonate buffer (pH 9.6) on Maxisorp microtiter plates (Nalge Nunc International). Peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories) was used as the conjugate at a 1:3,000 dilution, with o-phenylenediamine used as the substrate. Serum samples were diluted 1:100 in phosphate-buffered saline with 0.6 M NaCl, 1% skim milk, and 0.05% Tween 20 and tested in duplicate. The reaction was stopped with 1 M sulfuric acid when the optical density (OD) for a predefined HSV-2-positive serum sample reached a value of 2.0 ± 0.3. The absorbance was measured at 490 nm, and the results are given as the mean value for each duplicate. The cutoff value was defined as follows: a panel of 40 randomly selected serum samples which were negative by the ELISA with the HSV-1-derived type-common membrane antigen and the HPLmgG-2 antigen were analyzed by sgG-2 ELISA. A negative control serum sample (CS) with reactivity equal to the mean + 2 standard deviations (SDs) for the panel was included on each plate. A serum sample was considered negative if the OD value for the sample was lower than that for the CS plus 0.25 OD units. A serum sample was considered positive if the OD value for the sample was higher that that for the CS plus 0.35 OD units. Specimens with OD values greater than or equal to that for the CS plus 0.25 OD units and less than or equal to that for the CS plus 0.35 OD units were considered equivocal and were retested, and the result of the second test was used. Detection of HSV-2 IgM. An in-house indirect immunofluorescence technique was used to detect IgM antibodies against HSV-2 (HSV-2 IgM), as described previously (30). Commercially available assays. The serum samples in panels A to E were analyzed by an HSV-2-specific assay, the FOCUS2 assay (Focus Technologies). In addition, the sera in panel E were tested for HSV-1-specific antibodies by the HerpeSelect 1 ELISA (Focus Technologies). The assays, which are based on recombinant-produced gG-1 or gG-2 expressed in a baculovirus system, were performed according to the instructions of the manufacturer. Samples with equivocal results were retested, and the result of the second test was used. Statistics. Fisher's exact test was used for determination of P values. 　 RESULTS Top Abstract Introduction Materials and Methods Results Discussion References Reactivities by sgG-2 ELISA and FOCUS2 assay. The results for sera in panels A to C obtained by the sgG-2 ELISA and the FOCUS2 assay are shown in Fig. 1A and B, respectively. All 109 serum samples in panel A, which included sera from individuals with culture-proven HSV-1 infection and no detectable HSV-2 antibodies by WB, were negative by the sgG-2 ELISA, while 2 serum samples were positive by the FOCUS2 assay. Two of 106 serum samples from patients with a culture-proven HSV-2 infection (panel B) were consistently negative by the sgG-2 ELISA. In addition, three specimens from panel B showed equivocal reactivities but became positive on retesting. Thus, 104 (of 106) serum samples were considered positive by the sgG-2 ELISA. All sera in panel B (106 of 106 serum samples) were positive by the FOCUS2 assay. One serum sample in panel C, which included 100 serum samples with no detectable antibodies against HSV-1 and HSV-2, was positive by the sgG-2 ELISA. WB confirmed that this serum sample was HSV-2 negative. None of the sera in panel C was positive by the FOCUS2 assay. The results and the distribution of the sera according to the clinical diagnosis for sera containing IgM antibodies or ANA (panel D) are shown in Fig. 2. One serum sample (of 70 tested) was false positive by the sgG-2 ELISA, while 13 of the 70 serum samples were false positive by the FOCUS2 assay. Four of these 13 serum samples presented reactivities close to the index values specified as the cutoff for positive samples. fig.ommitted FIG. 1. Box plots showing the reactivities of sera by the sgG-2 ELISA (A) and the FOCUS2 assay (B). Panel A, HSV-1 positive and HSV-2 negative sera; panel B, HSV-2 positive sera; panel C, HSV-1- and HSV-2 negative sera. The boxes represent the 25th and 75th percentiles, and the whiskers represent the 10th and 90th percentiles. The median values are shown as horizontal lines. Dots outside the boxes indicate outliers. No upper outliers or median value are presented for the samples in panel B tested by the FOCUS2 assay, as calculated index values >6.5 were all given an index value of 6.5.

　


fig.ommitted FIG. 2. Outcomes of the sgG-2 ELISA and the FOCUS2 assay for 70 HSV-2-negative serum samples containing IgM antibodies against CMV, EBV, or M. pneumoniae or for sera with ANA.

　

Sensitivities and specificities. The sensitivities and specificities of the sgG-2 ELISA and the FOCUS2 assay, which were determined on the basis of the results for sera included in panels A to D, are summarized in Table 1. The overall sensitivity and specificity for the sgG-2 ELISA, as judged from the results presented for panels A to C, were 98 and 99.5%, respectively. The FOCUS2 assay showed a sensitivity of 100% and a specificity of 99%. The sgG-2 ELISA showed a significantly (P = 0.001) higher specificity (99%) than the FOCUS2 assay (81%) for the sera included in panel D (tricky sera).


fig.ommitted TABLE 1. Performance of the sgG-2 ELISA and the FOCUS2 assay

　

CVs of sgG-2 ELISA. An HSV-2-positive serum sample was tested at 28 positions (wells) in the same plate. The mean ± SD OD value was 2.26 ± 0.10, giving an intra-assay coefficient of variation (CV) of 4.4%. The interassay CV was determined by comparing the reactivities of the serum sample on 14 different occasions. The mean ± SD OD value was 1.95 ± 0.21, giving an interassay CV of 10.8%.

Seroconversion in patients presenting with a first episode of HSV-2-induced lesions. Consecutive serum samples from 21 patients (panel E) were analyzed by using four different HSV-2-specific IgG assays (the sgG-2 ELISA, the FOCUS2 assay, the HPLmgG-2 ELISA, and WB) and one assay for the detection of IgM antibodies. A positive culture was obtained from HSV-2 lesions on day 0. A serum sample drawn at the same time or within 7 days prior to or after day 0 was considered an acute-phase sample. The results are shown in Fig. 3. All acute-phase serum samples were HSV-2 negative by the sgG-2 ELISA. In total, 12 patients showed seroconversion; but the serum sample from 1 additional patient (patient 21) presented with an equivocal result, even though the sample was tested twice. Acute-phase serum samples from seven patients (patients 4, 6, 11 to 13, 18, and 20) were HSV-2 positive by the FOCUS2 assay. The consecutive serum samples from one patient (patient 4) were negative. The consecutive serum samples from 14 patients showed seroconversion. Thus, HSV-2 infection was detected by the FOCUS2 assay in all 21 patients. Fifteen patients showed seroconversion by the HPLmgG-2 ELISA. When sera were assayed by the WB technique, the acute-phase sera of 3 patients (patients 7, 12, and 20) were HSV-2 positive and 16 patients showed seroconversion in consecutive serum samples. Thus, HSV-2 infection was detected in 19 patients by the WB technique. IgM antibodies were detected in acute-phase serum samples or consecutive serum samples in 16 patients. Three patients were infected with HSV-1, as judged by the reactivity to the gG-1 antigen (Fig. 3).


fig.ommitted FIG. 3. Times to seroconversion for 21 patients (patients 1 to 21) presenting with a first episode of HSV-2-induced lesions and with no detectable IgG antibodies against HSV-2 in the acute-phase sera by the HPLmgG-2 ELISA. HSV-2 isolation was performed with samples obtained on day 0. For all patients except patients 2, 12, and 21, acute-phase sera were drawn at day 0. Sera were also assayed for HSV-2 IgG antibodies by WB, the sgG-2 ELISA, and the FOCUS2 assay. HSV-1 antibody status was evaluated by using a commercially available assay (HerpeSelect 1 ELISA). Patients with IgG antibodies against HSV-1 are indicated by asterisks. Results are denoted as positive (+) or negative (-). Equivocal results are denoted ±. IgM antibodies against HSV-2 (HSV-2 IgM) were detected by an indirect immunofluorescence technique and are presented as titer values. NA, sample not available.

　


　 DISCUSSION

Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Most of the envelope glycoproteins of HSV-1 and HSV-2 have extensive genetic similarities and induce a cross-reactive antibody response. Commercially available assays based on crude antigen preparations therefore present inaccurate results with low specificities (1). The purpose of the present study was to evaluate sgG-2 as a novel antigen in an ELISA format for type-specific serology and to compare the performance of the sgG-2 ELISA with that of a well-documented and commercially available assay based on recombinant-produced gG-2 (the FOCUS2 assay). We showed here that among sera collected from patients with culture-proven recurrent HSV-2-induced lesions (panel B), the sgG-2 ELISA had a sensitivity of 98%, results similar to those of the FOCUS2 assay (100%). The latter assay has previously been shown to be very sensitive compared to the results of WB (25). The specificities of the sgG-2 ELISA and the FOCUS2 assay were also high (>99%) for the sera included in panels A and C. In contrast, when tricky sera (panel D) were tested, the sgG-2 ELISA had a significantly lower number of false-positive results compared with the number obtained by the FOCUS2 assay. When the protocol for the sgG-2 ELISA was evaluated, we included 0.6 M sodium chloride in the incubation buffer to avoid nonspecific reactivity. This finding may be due to the fact that sgG-2 is a highly positively charged protein (pI 10.3). The recombinant-produced gG-2 antigen used in the FOCUS2 assay contains a truncated protein of 661 amino acids, including sgG-2. According to information provided by the manufacturer, the incubation buffer does not contain additional sodium chloride, which may explain the nonspecific reactivity for sera containing IgM antibodies. It is notable that nine of the samples with false-positive results by the FOCUS2 assay showed reactivities at the same levels as the low-positive control and would thereby be judged to be clearly positive. The other four serum samples showed relatively low levels of reactivity, with index values close to the cutoff value. A positive result judged on the basis of the results of the FOCUS2 assay should perhaps be interpreted with caution when sera from patients with acute viral or bacterial infections are analyzed.

Among the samples from patients presenting with a first episode of HSV-2-induced lesions, acute-phase sera from eight patients were HSV-2 positive by either the WB technique or the FOCUS2 assay. IgM antibodies are frequently detected after a primary HSV-2 infection (12, 31). Six of the eight patients developed IgM antibodies, a finding which suggests that the positive IgG reactivities of the acute-phase sera reflected an early seroconversion. However, it has been described previously that patients presenting with a first episode of HSV-2-induced lesions indeed have recurrent infections (22). Some of these eight patients may therefore represent recurrently infected individuals. The acute-phase serum sample from one patient (patient 4) was positive by the FOCUS2 assay, but a consecutive serum sample was negative. In other studies, this phenomenon has been described as "seroreversion" (14, 26). Sporadic reversal from positive reactivity to negative reactivity has been reported by use of the Gull gG-based HSV-2 ELISA. However, this was not related to a loss of WB profiles and most likely represents a fluctuation in the test itself (1). Nevertheless, among HSV-2-positive pregnant women, the loss of reactivity to mgG-2 by ELISA is a common finding during the end of pregnancy (9).

An aspect of relevance to the performance of a type-specific assay is the period of time from the time of transmission of HSV-2 to the time when antibodies are initially detected in the host. Although few serum samples were included in panel E, our results suggest that after HSV-2 infection antibodies against mgG-2 are detected earlier than antibodies against sgG-2. The anti-mgG-2 antibodies recognize linear epitopes of the protein to a high degree (21). WB can therefore be used as a sensitive and specific method for confirmation of the presence of type-specific anti-gG-2 antibodies. In contrast, anti-sgG-2 antibodies identify mostly nonlinear epitopes, which by WB have no reactivity to virus-infected cell lysates or purified sgG-2 antigen prepared under reducing conditions (18). This finding makes WB unsuitable as an alternative and sensitive method for the detection of anti-sgG-2 antibodies. The observed delayed antibody response to sgG-2 may therefore be explained by a lower sensitivity of the sgG-2 ELISA compared to those of assays based on mgG-2 or whole gG-2 antigens. One limitation of the present study is that all HSV-2-positive serum samples examined were drawn from patients presenting with HSV-2-induced lesions. Thus, sgG-2 must be further evaluated in clinical settings with sera from patients with asymptomatic HSV-2 infection.

It is crucial that an assay have a high degree of specificity to obtain a high predictive value of positive results. False-positive results lead to the provision of incorrect information to individuals and couples, which may cause psychological distress (23). In clinical settings, one strategy to increase the accuracy of an HSV-2-specific assay would be to confirm the results for positive samples by another assay. The WB technique is not suitable for routine diagnostic purposes, as the method is laborious and complex to perform and its results are difficult to interpret. The sgG-2 protein represents a novel antigen which, when used in an ELISA format, offers a high degree of specificity. sgG-2 can therefore be used as an additional antigen for the detection of type-specific IgG antibodies against HSV-2.

　


　 ACKNOWLEDGMENTS

This work was supported by grants from Local Research and Development Council of Göteborg and Southern Bohuslän (grant VGFOUGSB-172) and the Swedish Society of Medicine.

We thank Inga-Lill Hulthén for skillful technical assistance.



　 REFERENCES

日期：2007年5月10日 - 来自[2003年第41卷第8期]栏目
螺旋藻多糖对HSV-2糖蛋白gB mRNA表达的抑制作用

　　【摘要】 目的 探讨钝顶螺旋藻多糖(PSP)对HSV-2糖蛋白gB mRNA表达的影响及其机制，为进一步开发该药提供理论依据。方法 以不同剂量的PSP作用于HSV-2感染的Vero细胞，应用地高辛标记寡核苷酸探针，检测HSV-2糖蛋白gB mRNA的表达。结果 PSP作用组HSV-2糖蛋白gB mRNA的表达显著低于阳性对照组(P＜0.01)。结论 PSP抗HSV-2作用的机制之一为抑制HSV-2糖蛋白gB基因的转录。

　　【关键词】 螺旋藻多糖；单纯疱疹病毒2型；原位杂交

　　　Inhibition of HSV-2 glycoprotein gB mRNA expression by polysaccharide from spirulina platensis(PSP)

　　ZHOU Xiao-dong,

YU Hong.Qingdao Stomatological Hospital, Qingdao 266003,China

　　【Abstract】 Objective To explore the effects and mechanism of PSP on the expression of HSV-2 gB mRNA and to provide a theoretical support for developing new drugs.Methods PSP was applied at various concentrations to Vero cells infected by HSV-2, In situ hybrization was adopted to explore the expression of HSV-2 gB mRNA with digoxigenin(DIG)-labeled HSV-2 oligoprobe. Results HSV-2 gB mRNA were less expressed in PSP treated group than that in the control (P＜0.01).Conclusion One of the mechanism of PSP against HSV-2 may be explained by the considerable inhibition of HSV-2 gB gene trscription.

　　【Key words】 polysaccharide from spirulina platensis; HSV-2; in situ hybrization

　　单纯疱疹病毒2型(herpes simplex virus-2, HSV-2)在人群中感染较为普遍。孕妇生殖器感染可引起早产、流产、死胎、胎儿畸形以及智力低下等，并与子宫颈癌的发生密切相关［1，2］。近几年研究表明：钝顶螺旋藻多糖（polysaccharides from Spirulina platensis，PSP）是钝顶螺旋藻中具有抗肿瘤、抗辐射、抗病毒和免疫调节作用的重要生物活性物质［3，4］。我们的前期实验结果表明，PSP有抗HSV-1的作用，但PSP抗HSV-2的实验研究未见报道。本文研究了PSP对HSV-2糖蛋白gB mRNA表达的抑制作用，结果报告如下。

　　1 材料与方法

　　1.1 药品与试剂配制 螺旋藻多糖：参照文献［5，6］，采用改良的三氯乙酸去蛋白法提取螺旋藻多糖，纯度达92％以上。以双蒸水配成10mg/ml母液，过滤除菌，分装，4℃保存，临用时以DMEM培养基稀释至所需浓度。 细胞培养所用的DMEM干粉培养基为美国GIBCO公司产品，胰酶为Amersco公司产品，新生牛血清为杭州四季青公司产品；地高辛DNA检测试剂盒为Roche公司产品。

　　1.2 细胞株与病毒 非洲绿猴肾细胞Vero细胞株为本室保存传代细胞系。于DMEM基础培养液中加入10%的新生牛血清，100U/ml青霉素，100μg/ml链霉素，置5％CO2孵箱中37℃培养，3天传代1次。标准单纯疱疹病毒2型（HSV-2）333株来源于中国协和医科大学，Vero细胞中传代，毒种保存于-70℃。

　　1.3 病毒滴度的测定 以TCID50表示病毒毒力。取上述制备好的毒株，做10倍系列稀释，10-1至10-10。将各稀释度的毒株接种于Vero细胞，各6复孔，同时设阴性对照。37℃培养3天后，观察细胞病变，以出现细胞病变的最高稀释倍数的倒数作为TCID50。

　　1.4 原位杂交检测HSV-2糖蛋白gB mRNA的表达

　　1.4.1 探针的设计［7，8］ 根据Gen Bank中HSV-2 全基因序列，设计对应于HSV-2糖蛋白gB基因序列的寡核苷酸探针序列为5′-ggcgactttgtgtacatgtccccgttttacggctaccgg -3′，由上海生工公司合成，并于5′末端标记地高辛。

　　1.4.2 原位杂交［9］ Vero细胞制成2×105/ml细胞悬液，接种在放有盖玻片（飞片）的培养板中，每孔加入1.8ml细胞，于5％CO2孵箱中37℃培养24～48h，弃生长液，加约100 TCID50病毒液50μl，37℃吸附2h，吸出病毒液，加200μl不同浓度的PSP（100μg/ml、50μg/ml和10μg/ml）， 置5％CO2孵箱中37℃培养。24h后，取出细胞飞片经4%多聚甲醛室温下固定20min，PBS漂洗2次，梯度乙醇脱水，蛋白酶K（1μg/ml）37℃消化30min，PBS漂洗2次，2×SSC洗2次，梯度乙醇脱水，空气干燥。每张飞片滴加50μl 杂交液，预杂交2h；加入含探针的杂交液，置于密闭湿盒中，60℃过夜。杂交结束后，飞片在室温下依次经2×SSC、1×SSC、0.5×SSC漂洗各2次，于BufferⅠ洗液中10min，封闭液BufferⅡ中37℃孵育30min，加入抗体液37℃孵育30～60min，BufferⅠ洗液中10min，置BufferⅢ平衡10min，新鲜配制的显色液中避光孵育10min至数小时，终止反应。中性树胶封片。

　　实验同时设3个阴性对照：(1) 杂交前用Rnase A 100μg/ml预处理细胞飞片，37℃，1h；(2) 加未标记的探针进行反应；(3) 正常的Vero细胞。阳性对照为未加PSP作用的HSV-2感染Vero细胞组。所有操作步骤与其他样本相同。结果判定：400×光镜下随机选择视野，观察200个细胞，计数杂交阳性的细胞数。阳性信号呈明亮的蓝紫色。

　　1.5 统计学方法 核酸杂交结果以(x±s)表示，多组数据比较采用方差分析。

　　2 结果

　　光镜下观察原位杂交结果，可见表达HSV-2糖蛋白gB基因的Vero细胞被染成蓝紫色，染色部位位于细胞浆，而正常Vero细胞未见阳性信号表达（图1）。不同剂量PSP作用的HSV-2感染组，其糖蛋白gB mRNA的表达显著低于阳性对照组（P＜0.01），10μg/ml、50μg/ml、100μg/ml PSP可使gB mRNA的表达由86.61%分别降至62.29%、46.12%和31.56%（表1）表1 PSP对HSV-2糖蛋白gB mRNA表达的影响

　　3 讨论

　　目前临床应用的抗疱疹病毒药物主要为核苷类衍生物，多通过单一环节抑制病毒复制，如作用于病毒的DNA合成酶，它们以酶反应底物类似物的形式与正常底物竞争，掺入病毒核酸链，造成病毒核酸复制中断，发挥抑制病毒作用，但易产生耐药性、副作用大［10，11］。因此研究作用于病毒基因复制、转录及转译等多环节的药物和制剂是当今国内外开发抗病毒药物的热点。

　　我们的前期研究工作发现，PSQ对Vero细胞无明显的毒性，可明显阻滞HSV-2向细胞吸附，并有效地抑制病毒复制，但不影响病毒的释放。HSV-2为双股DNA病毒，其复制是一个相当复杂但又十分程序化的过程。已知HSV-2包膜糖蛋白gB是与特异性细胞受体相互作用的病毒配体分子，与病毒吸附有关［1，12］。原位杂交结果表明不同剂量PSP作用的HSV-2感染组，其包膜糖蛋白gB mRNA的表达显著低于阳性对照组（P＜0.01），10μg/ml、50μg/ml、100μg/ml PSP可使gB mRNA的表达由86.61%分别降至62.29%、46.12%和31.56%，进一步从分子水平直接证明PSP可抑制HSV-2病毒的吸附。

　　对一些病毒病的治疗，国内外学者主张早期、联合用药，尤其是作用机制不同的药物联合应用，可提高疗效、降低毒副作用。由于PSP为天然化合物，具有无毒、高效的特点，明显不同于常用抗病毒药，同时兼有抗病毒和免疫调节双重作用，在发展新型抗病毒药物中具有十分重要的意义，也是合成药所不具有的优点。因此，PSP作为一种高效低毒的抗病毒药物，值得进一步研究和开发。

　　【参考文献】

　　1 闻玉梅. 现代医学微生物学.上海: 上海医科大学出版社, 1999，1227-1244.

　　2 Whitely RJ, Roizman B. Herpes simplex virus infections. Lancet，2001, 357:1513-1518.

　　3 涂芳, 杨芳, 郑文杰，等. 螺旋藻多糖的研究进展. 天然产物研究与开发, 2005, 17（1）: 115-119.

　　4 于红，张学成. 螺旋藻多糖抗HSV-1作用的体外实验研究. 高技术通讯,2002,12(9):65-69.

　　5 Lee JB, Hayashi T, Hayashi K, et al. Further purification and structural analysis of calcium spirulan from spirulina platensis. J Nat Prod, 1998, 61(9):1101-1104.

　　6 章银良，李红旗，高峻，等.螺旋藻多糖提取新工艺的研究. 食品与发酵工业, 1999，25(2)：13-18.

　　7 Cone, RW, Hobson AC, Palmer J, et al. Extended duration of herpes simplex virus DNA in genital lesions detected by the polymerase chain reaction. J Infect Dis，1991, 164:757-760.

　　8 Dolan A, Fiona E. The genome sequence of herpes simplex virus Type 2. J Virol, 1998, 72(3): 2010-2021.

　　9 卢圣栋. 现代分子生物学实验技术. 第二版. 北京: 中国协和医科大学出版社, 1999，195-229.

　　10 Naescens L, De Clercq E. Recent developments in herpes virus therapy. Herpes, 2001，8(1):12-16.

　　11 Coen DM. Antiviral drug resistance in herpes simplex virus. Adv Exp Med Biol，1996, 394:49-57.

　　12 Trybala E, Liljeqvist JA, Svennerholm B, et al. Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate. J Virol, 2000,74(19): 9106-9114.

　　 *基金项目：青岛市科技计划项目（编号：02-2-kj-yi-31）

　　作者单位： 1 266003 山东青岛，青岛市口腔医院

　　2 266021 山东青岛，青岛大学医学院微生物学教研室

(编辑：悦 铭)
日期：2006年7月19日 - 来自[2006年第7卷第4期]栏目
第二节基因结构

第二节　基因结构

　　一、基因组结构

　　HSV-2基因组为双链线性DNA分子，碱基组成含69%G+C，分子量为96×106D。根据基因组两个通过共价键相连的分别被称为L（长）和S（短）的组分的方位不同，HSV-2DNA可有四种异构体。L组分包括82%的病毒DNA，并且有一独特序列“U”，其两侧有一反转重复区域ab或b′a′，分别含6%病毒DNA。S组分占18%的病毒DNA，也有一独特序列（US），其两侧亦有反转重复序列a′c′和ca。a′c′和ca分别含4.3%DNA。彼此相关的L和S序列的倒置衍生于位点特异性重组，重组是由末端“a”序列区的病毒基因产物介导的。HSV-2DNA两端可连接成环状，此结构与病毒的致癌与潜伏有关。HSV-2DNA中L和S的末端重复序列主要编码特定的即刻早期转录产物，而独特序列主要编码特定的早期及晚期多肽和糖蛋白。

　　HSV-2有三类基因，即α、β、γ。其中α基因在感染中最早表达且不需要前病毒蛋白的合成；β基因的感染需要α蛋白的预先合成；γ基因的表达需要病毒DNA的复制。现已证实HSVmRNA可合成三类基本蛋白：急早期（immediate early，α）、早期（early，β）和晚期（late，γ）蛋白，β蛋白在DNA复制中起作用，包括胸苷激酶，DNA多聚酶及大多数DNA结合蛋白，而大部分糖蛋白则多为γ蛋白。HSV-2蛋白合成呈连锁调节，即α基因产物诱导β基因表达，而β基因产物（加上一种α蛋白ICOP4）反过来又可终止α基因表达，同时诱导γ基因的表达，γ基因产物则可负调节β基因的表达并且可作为下一轮病毒复制合成α蛋白的起始信号。

　　迄今为止，已有6个编码糖蛋白的基因已定位于HSV-2线性位点上，其基因产物分别被命名为gB、gC、gD、gE、gG和gH，编码gB、gC的基因位于UL区，而编码gD、gE、gG和gH的US区。

　　二、与诱导细胞转化有关的基因定位

　　Aurelian对所有实验中HSV-2感染的细胞株（包括肿癌细胞株）作了分析，发现病毒DNA共有的序列仅两处，一处是Bg1ⅡG片段，位于0.21-0.33图单位,该DNA序列与感染细胞在低血清培养剂中的增殖有关,而与病毒致癌无关;另一处是Bg1ⅡC片段,位于0.45～0.58图单位,能诱导细胞转化.HSV-2诱导仓鼠胚胎成纤维细胞转化的DNA区域位于Bg1Ⅱ/Hpal-CD片段,相当于0.44～0.52图单位.HSV-2DNA的Bb1ⅡN片段位于0.58～0.63图单位,为形态学转化区,已知0.58～0.628中无特定的编码功能.L+K-细胞株或3T3tk-细胞株能通过HSV-2感染转化成TK+表型,导致转化所需的HSV-2DNA序列位于0.28-0.32图单位。
日期：2006年1月15日 - 来自[基因诊断与性传播疾病]栏目
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

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