生殖器疱疹病毒感染检测方法的临床应用

目的 :　评价检测生殖器疱疹病毒实验方法的临床应用价值。方法 :　同时采用细胞培养法、定量PCR法、间接免疫荧光法 (IIF)对 5 3例生殖器疱疹 (GH)感染患者进行了HSV检测。结果 :　细胞培养、定量PCR检测HSV阳性率明显高于IIF法 (P <0 .0 1) ,细胞培养与定量PCR比较无显著性差异 (P >0 .0 5 )。三种方法检测GH患者皮疹水疱内的阳性率无明显差异 (P >0 .0 5 )。IIF法检测糜烂结痂的阳性率仅为 2 8.6 %。结论 :　三种方法均适用GH水疱期患者标本的检查。而定量PCR、细胞培养检测GH糜烂结痂患者标本较IIF法敏感     

Diagnosis of genital herpes by real time PCR in routine clinical practice

BACKGROUND: Virus isolation in cell culture is the recognised diagnostic gold standard for genital herpes. Although increasing evidence indicates that polymerase chain reaction (PCR) provides a more rapid and sensitive diagnostic method, its implementation in routine diagnostic settings has been limited by concerns over contamination and cost. OBJECTIVE: To evaluate the feasibility of replacing virus culture with PCR for the diagnosis of genital herpes in settings serving large populations of genitourinary medicine (GUM) attendees. METHODS: Genital swabs collected from 233 consecutive GUM attendees with suspected genital herpes were tested in parallel by virus culture and automated real time PCR. Three specimen preparation methods were evaluated and the assay reliability was assessed by repeat testing, comparison with a commercially available assay, and herpes simplex virus (HSV) sequence analysis. Probe melting temperatures (Tm) were used to differentiate between HSV types without additional post-PCR steps. RESULTS: HSV was detected in 79/233 (34%) samples by virus culture and 132/233 (57%) samples by PCR. PCR significantly increased HSV detection in both early (< 5 days) and late (> or = 5 days) presentations and in both first and recurrent episodes. HSV detection and typing by PCR was achieved within less than 4 hours leading to a significant reduction in labour compared to virus culture. Most specimens (120/132, 91%) were typed as HSV-2. Results were highly reproducible. CONCLUSIONS: Real time PCR is a highly reproducible, rapid, and labour efficient method for HSV detection in genital swabs. Its implementation is feasible in routine diagnostic settings.     

生殖器溃疡中单纯疱疹病毒的检测和分型

目的：了解性病门诊生殖器溃疡患者中单纯疱疹病毒（HSV）感染情况，并评价聚合酶链反应（PCR）－微孔板反向杂交检测和分型方法在生器疱疹诊断中的意义。方法：采用病毒分离培养、普通PCR和PCR－微孔板反向杂交法同时对200份生殖器溃疡标本作了HSV检测与分型。结果：PCR－微孔板反向杂交法的敏感性和特异性分别为98．1％和95．9％，PCR－微孔板杂交法分型结果与病毒分离培养法和普遍PCR的分型结果完全相符。生殖器溃疡中HSV检出率为30％（60／200），其中HSV－2感染占96．7％（58／60）。结论：HSV－2是性病门诊患者生殖器溃疡的主要病因之一，PCR－微孔板反向杂交法是一种适用生殖器溃疡标本中HSV的检测与分型的快速、敏感和特异的诊断方法。     

Routine detection of herpes simplex virus and varicella zoster virus by polymerase chain reaction reveals that initial herpes zoster is frequently misdiagnosed as herpes simplex

The differential diagnosis of herpes simplex and zoster may require virological confirmation, yet virus typing is not regarded as necessary in routine dermatological assessment. In an attempt to evaluate the clinical benefits of the routine detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), we analysed skin swabs from 110 patients who were diagnosed at the first clinical visit as having herpes simplex ( n  = 45) or zoster ( n  = 65). Viruses were typed using the polymerase chain reaction (PCR) with the general primer pair GPHV-RU. PCR analysis showed that at the initial clinical presentation, herpes simplex in these patients was not mistaken for zoster but that zoster was incorrectly diagnosed as herpes simplex in nine cases. Thus these results suggest that initial zoster often mimics herpes simplex, hence routine PCR diagnosis of HSV and VZV or alternative rapid diagnostic approaches may be beneficial in these cases.     

定量PCR和间接免疫荧光法联合检测生殖器疱疹病毒感染

目的：研究间接免疫荧光法（IIF）检测生殖器HSV感染的敏感性和特异性。方法：以定量PCR为对照,用HSV型共同性糖蛋白单克隆抗体为夹心的IIF法,检测了94例临床诊断为生殖器疱疹的患者皮疹中的HSV。结果：IIF法检测HSV的敏感性为74．12％,特异笥为55．60％;总阳性率（71．30％）,明显低于定量PCR法的阳性率（90．40％）（P＜0．05）,但两种方法检测GH患者皮水疱内的HSV阳性率无明显差异（86．20％vers.97.00%),而检测糜烂性皮疹内的HSV时,PCR法的阳性率高于IIF法（P＜0．05）,结论：IIF法具有简单,快捷的优点,适用于检测早期可疑GH患者皮疹内HSV,有临床实用价值。     

Polymerase chain reaction for diagnosis of genital herpes in a genitourinary medicine clinic

BACKGROUND: Polymerase chain reaction (PCR) has well established advantages over culture for diagnosis of herpes viruses, but its technical complexity has limited its widespread application. However, recent methodological advances have rendered PCR more applicable to routine practice. Aim: To compare automated PCR with viral culture for diagnosis of genital herpes. METHODS: We studied 236 patients presenting with clinical features suggestive of genital herpes at an inner city genitourinary medicine clinic. Two swabs were taken from each patient. Cell culture and typing were performed by standard methods. Automated PCR was performed using the LightCycler instrument and the infecting viral type was determined by restriction endonuclease digestion of amplicons. RESULTS: 109 patients (46%) had a positive test for herpes simplex virus (HSV). In 88, both PCR and culture were positive; in 21 PCR only was positive. With both detection methods, lesion duration and morphology were associated with HSV detection. Compared with culture alone, use of PCR increased sensitivity by 13.3% in specimens from vesicular lesions, by 27.4% from ulcerative lesions, and by 20.0% from crusting lesions. CONCLUSIONS: We advocate adoption of automated PCR as an efficient HSV detection and typing method for diagnosis of genital herpes in routine clinical practice. PCR allowed rapid laboratory confirmation of the diagnosis and increased the overall HSV detection rate by 24%.     

National survey of diagnostic services for genital herpes

OBJECTIVE: To investigate the provision of diagnostic services for genital herpes simplex virus (HSV) infection in the United Kingdom. METHODS: National survey of laboratories providing diagnostic services for genital herpes. RESULTS: Completed questionnaires were returned from 25/32 (78%) laboratories participating in the Clinical Virology Network, including seven in London, 12 in the rest of England, one in Wales, four in Scotland, and one in Northern Ireland. Virus culture was the diagnostic method of choice in 20/25 (80%) laboratories; 5/25 (20%) routinely used HSV DNA detection by polymerase chain reaction (PCR). HSV PCR for DNA detection in cerebrospinal fluid (CSF) was available in 17/25 (68%) laboratories. Typing of isolates (HSV-1 or HSV-2) was performed routinely in 22/25 (88%) laboratories. Only 2/25 (8%) laboratories offered HSV type specific serology, although an additional 12/25 (48%) referred requests elsewhere. Consistent with this finding, the number of HSV type specific antibody tests referred to the Health Protection Agency increased by nearly fivefold between 1997 and 2003. CONCLUSIONS: Virus culture remains the preferred diagnostic method for genital herpes, despite evidence indicating that its sensitivity is suboptimal compared to PCR. As HSV PCR is widely available for testing of CSF, it is recommended that clinicians and virologists discuss ways to implement PCR testing of genital swabs, thus enabling greater diagnostic accuracy. A call is made for studies to assess the use of HSV type specific serology in genitourinary medicine (GUM) settings, now that rapid and validated assays have become available and guidelines have been issues to provide recommendations on their use.     

多聚酶链反应和病毒培养法联合研究生殖器疱疹病毒感染

采用多聚酶链反应 (PCR)和猴肾细胞 (Vero)病毒培养法同时检测了临床诊断为GH的 3 0例患者 ,分析了两种方法检测HSV的总阳性率 ,并就发病后不同时期取材HSV阳性率进行了分析 ,还用PCR法对HSV进行了分型检测 ,表明PCR法检测HSV较病毒培养法更快速、敏感 ,且能对HSV进行分型 ,适于临床使用     

Detection of herpes simplex virus DNA in the peripheral blood during acute recurrent herpes labialis.

BACKGROUND: Although herpes simplex virus (HSV) has been detected in the peripheral blood of immunocompromised patients and in neonates with disseminated disease, the extent to which this virus may be present in the blood during a localized infection in otherwise healthy adults is unknown. OBJECTIVE: The purpose of this study was to determine whether HSV may be detected in the peripheral blood during acute recurrent herpes labialis. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from otherwise healthy adults with recurrent herpes labialis, both during an acute episode and several weeks after the lesions had healed. The PBMCs were examined for the presence of HSV with the polymerase chain reaction (PCR) and viral culture. RESULTS: By PCR, HSV DNA was detected in 7 of 34 specimens from an acute episode but in none of 24 specimens in the convalescent stage (p less than 0.004). PBMCs from seven donors, who were seronegative for HSV, were also negative for HSV by PCR. Viral cultures of 22 PBMC specimens were negative (including four specimens that were positive by PCR). CONCLUSION: The presence of HSV DNA in the blood is a transient phenomenon limited to the period of active infection in a minority of patients with herpes labialis, although it may be important in the development of disseminated disease as well as in the pathogenesis of herpes-associated cutaneous processes such as erythema multiforme.     

Duplex real-time polymerase chain reaction assay for detection and quantification of herpes simplex virus type 1 and herpes simplex virus type 2 in genital and cutaneous lesions

BACKGROUND: A sensitive and specific method for detecting herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) is important for diagnosing genital and cutaneous infections. GOAL: The goal of this study was to compare quantitative real-time polymerase chain reaction (qPCR) with virus culture for diagnosis of genital and cutaneous HSV-1 and HSV-2. STUDY DESIGN: A duplex qPCR system for quantification of DNA from HSV-1 and HSV-2 was developed. Duplicate swabs for PCR and virus culture were collected from 89 patients attending our sexually transmitted infection and dermatology clinic. RESULTS: The duplex qPCR had a linear measure interval of 10-10 copies/mL. The detection limit was between 1 and 5 copies per reaction. qPCR detected HSV in 57 (64%) specimens and virus was isolated in 45 (50%) cases. First-episode infections showed higher viral quantities with a median value of 4.2 x 10 copies per reaction compared with recurrent infections with 1.0 x 10 (P = 0.0002). HSV-1 was more likely to be the cause of first-episode genital infections (72%), and HSV-2 of recurrent and atypical genital manifestations (73%). CONCLUSION: Real-time PCR is a sensitive method for diagnosing genital herpes, and the duplex format is convenient for typing. The method increased the detection rate by 27% compared with virus culture.     

Rapid diagnosis in varicella and herpes zoster: re-evaluation of direct smear (Tzanck test) and electron microscopy including colloidal gold immuno-electron microscopy in comparison with virus isolation

The Tzanck test and electron microscopy with the technique of colloidal gold labelling in varicella-zoster virus (VZV) infections were compared with virus isolation in 54 patients with clinically suspected varicella or herpes zoster infection. The Tzanck test and direct electron microscopy can determine whether or not an eruption is herpetic but cannot distinguish between herpes simplex virus (HSV) and VZV infection. However, colloidal gold immuno-electron microscopy, using monoclonal antibodies against HSV and anti-VZV IgG, can distinguish between these two herpes viruses. This achieves the same specificity as virus isolation followed by virus neutralization or virus typing using immunofluorescence techniques. The Tzanck test was positive in 91%, virus isolation, under optimal conditions of sampling and transportation, in 80%, direct electron microscopy (negative staining) in 80%, and colloidal gold immuno-electron microscopy after a virus concentration procedure in 95% of the cases. The colloidal gold technique offers a rapid diagnosis in patients with suspected VZV infection.     

间接免疫荧光法检测生殖器疱疹病毒

目的:评价间接免疫荧光试验(IFA)在生殖器疱疹(GH)诊断中的应用价值。方法:采用以单纯疱疹病毒(HSV)型共同性单克隆抗体为夹心的IFA法,检测了120例临床诊断为GH患者皮疹中的HSV,并与病毒培养法进行比较。结果:IFA检测HSV的总阳性率为85.8%,高于病毒培养法的阳性率(70.8%,χ2=12.04,P<0.01)。两种方法检测GH水疱内的HSV阳性率分别为93.3%和90.0%,无明显差异(χ2=1.96,P>0.05);而检测糜烂和结痂性皮疹内的HSV时,IFA的阳性率分别为92.6%和69.4%,均分别高于病毒培养法(75.9%,χ2=5.82,P<0.05;47.2%,χ2=14.17,P<0.01)。结论:IFA法具有简单、快速、敏感性高的优点,适于检测GH患者皮疹内HSV,有临床实用价值。     

Detection of cutaneous herpes simplex virus infections by immunofluorescence vs. PCR

BACKGROUND: Detection of cutaneous infections with herpes simplex virus (HSV) has proven difficult, as serum antibody tests sometimes are not sensitive and specific enough for that purpose. OBJECTIVE: This study was conducted to compare the sensitivity for detection of HSV of an immunofluorescence method (Syva Microtrak) and an internally controlled PCR. METHODS: Cutaneous swabs from skin lesions were analysed by immunofluorescence separately for HSV types 1 and 2 and by competitive PCR. Detection of PCR products was done by ELISA, if positive additionally by agarose gel electrophoresis. RESULTS: Of 79 samples 34 were PCR-positive by ELISA (34 = 100%), of which 23 (68%) were also positive on the agarose gel. Eleven samples (32%) were positive by immunofluorescence. No sample was positive by immunofluorescence and negative by PCR. CONCLUSIONS: These results demonstrate that immunofluorescence using Syva Microtrak is not suitable for exclusion of herpes simplex virus infection as sensitivity was only 32%. However, as immunofluorescence is cheaper and faster than PCR, first screening can be done with immunofluorescence, and negative samples can be investigated by PCR to finally prove or exclude the presence of HSV DNA.     

Detection of herpes simplex virus DNA in serum and oral secretions during acute recurrent herpes labialis

Although herpes simplex virus (HSV) has been detected in the peripheral blood of immunocompromised patients and in neonates with disseminated disease, the extent to which the virus may be present in the blood during a localized infection in otherwise healthy patients is still unknown. Literature on patterns of HSV shedding into the oral cavity at the prodromal stage of the disease, during recurrences, and also during asymptomatic periods is still lacking. The present study aims at the detection of HSV DNA in the serum and oral secretions during acute herpes labialis using a highly sensitive technique, the polymerase chain reaction (PCR). The study included 10 patients with acute herpes labialis and five healthy controls. Using PCR, herpes simplex virus DNA was detected in the serum of seven patients (70%) and in the saliva of nine patients (90%). One of the control cases showed positive HSV DNA in the saliva (20%). There was good statistical agreement between the presence of HSV DNA in the serum and saliva. Frequency of attacks, patient's age, and gender had no statistically significant effect on the presence of the virus in serum or in saliva. It is concluded that HSV viremia during attacks of recurrent herpes simplex is more frequent than previously appreciated.     

抗原捕获聚合酶链反应分型检测妇女生殖器单纯疱疹病毒

目的 :　建立直接分型检测妇女生殖器单纯疱疹病毒 (HSV)的抗原捕获聚合酶链反应 (AC -PCR)。方法 :　用抗HSV型共同性糖蛋白单克隆抗体 ,包被聚苯乙烯离心管 ,捕获HSV ,同时加入 3个引物 :HSV - 1 HSV - 2型共同性上游引物及HSV - 1和HSV - 2型特异性下游引物 ,进行PCR扩增。结果 :HSV - 1和HSV - 2标准病毒株均分别扩增出与设计大小相符的 4 77bp和 399bpDNA条带。AC -PCR可检测到 10PFUHSV - 1和 1PFUHSV - 2。用AC -PCR检测了 36 5份妇女生殖器拭子标本 ,阳性 10 1例(2 7.7% ) ,2 3例为HSV - 1(占 2 2 .8% ) ,78例为HSV - 2 (占 77.2 % ) ;其中 112份标本同时用AC -PCR和分离培养法检测 ,AC -PCR的阳性率为 2 6 .8% (30 112 ) ,分离培养法的阳性率为 2 0 .5 % (2 3 112 ) ,两者差异有显著性 (χ2 =4 .5 ,P <0 .0 5 )。结论 :　AC -PCR是特异、敏感、快速分型检测妇女生殖器HSV感染的方法     

酶联免疫吸附试验在检测生殖器疱疹病毒抗原中的应用

目的探讨酶联免疫吸附试验(ELISA)检测泌尿生殖道分泌物及皮损中单纯疱疹病毒(HSV)抗原在临床中的应用价值.方法用聚合酶链反应(PCR)和ELISA方法检测泌尿生殖道及皮损中HSV DNA和HSV抗原,并对两种方法的检测结果进行比较.结果118例标本中,PCR检出41例阳性,ELISA 42例阳性.在118例标本中,111例两种方法结果相符,7例不相符合.PCR阳性的41例中,ELISA阳性38例(敏感性92.68%);PCR阴性的77例标本中,ELISA阳性4例(特异性94.81%).结论ELISA检测HSV抗原的方法可直接检测出泌尿生殖道及皮损中HSV病原体,从而为生殖器疱疹诊断提供准确的实验依据,其敏感性、特异性高,方便,快速,值得临床推广应用.     

High frequency of detection of herpes simplex virus DNA in the oral cavity of patients with eczema herpeticum

BACKGROUND: It has been reported that herpes simplex virus (HSV) DNA was detected in the oral cavity of patients with herpes labialis under various conditions such as during oral surgery. OBJECTIVE: The frequency of detection of oral HSV DNA was compared between first or recurrent episodes of eczema herpeticum and recurrent type herpes labialis. PATIENTS AND METHODS: Oral swabs were collected from 7 patients with eczema herpeticum and 9 with herpes labialis. The detection of oral HSV DNA was performed by the polymerase chain reaction method. RESULTS: Oral HSV DNA was detected in 6 out of 7 patients (86%) with eczema herpeticum and 3 of 9 (33%) with herpes labialis. CONCLUSIONS: The high frequency of oral HSV DNA detection in eczema herpeticum suggests that subclinical herpetic lesions may develop in the oral cavity of patients with a first episode of eczema herpeticum or may occur during asymptomatic oral HSV shedding in people with recurrent eczema herpeticum.     

Premarket evaluation of the POCkit HSV-2 type-specific serologic test in culture-documented cases of genital herpes simplex virus type 2 [see comment

BACKGROUND AND OBJECTIVES: The genital herpes epidemic continues, in part, because patients with subclinical or atypical presentations cannot be identified by most herpes simplex virus (HSV) antibody tests. A new product, POCkit HSV-2, has been developed to rapidly and accurately detect antibodies to HSV type 2 (HSV-2) in capillary blood or serum. GOAL: Sera from patients with culture-documented genital or oral herpes were tested to determine the sensitivity and specificity of the POCkit HSV-2 rapid point-of-care antibody test (Diagnology, Belfast, Northern Ireland). STUDY DESIGN: Sera from 50 patients with culture-documented HSV type 1 (9 oral, 41 genital) and from 253 patients with genital HSV-2 were tested by POCkit HSV-2 for HSV-2 antibodies. Each subject had a positive culture for HSV within 6 months of serum collection. Sera were preselected to include only those that were seropositive to the respective virus subtype by University of Washington Western blot. RESULTS: Compared with viral culture and Western blot analysis, sensitivity of the POCkit HSV-2 test for HSV-2 antibody was 96%; specificity was 98%. CONCLUSION: This test provides rapid, accurate identification of HSV-2 antibody in subjects with established HSV infections.     

对ELISA检测生殖器疱疹患者HSV及其临床应用的评价

目的评价酶联免疫吸附试验(ELISA)检测生殖器疱疹病毒的临床应用价值。方法采用ELISA和分型聚合酶链反应(分型PCR)检测生殖器标本中的单纯疱疹病毒(HSV),两种试验结果不符合者采用不分型PCR检测。结果164例受检者中,ELISA法HSV阳性96例(58.5%),其中具典型皮损者阳性84例(80.8%,84/104),非典型皮损阳性12例(20.0%,12/60);分型PCRHSV阳性98例(59.8%),其中典型皮损者HSV阳性86例(82.7%,86/104),非典型皮损者阳性12例(20.0%,12/60)。HSV1感染者占生殖器疱疹的5.1%,HSV2感染占88.7%,HSV1和HSV2混合感染者占6.1%。ELISA的敏感性和特异性分别为96.7%和94.0%。结论ELISA检测HSV感染,其敏感性高、特异性强,方便、快速,尤其适合大批量样本的检测。     

定量PCR对生殖器疱疹患者尿道隐性HSV排毒研究

目的:研究GH发作期和间歇期患者泌尿生殖道隐性HSV-2排毒情况和沙眼衣原体(Ct)、解脲脲原体(Uu)阴性的非淋菌性尿道炎(NGU)患者HSV-2感染情况。方法:发作期GH患者分别取皮疹和尿道拭子标本;间歇期GH患者每周1次取尿道拭子共4次,正常对照组及Ct、Uu阴性的NGU患者取尿道拭子标本,用定量PCR法检测标本中的HSV-2DNA。结果:GH、NGU患者及正常组之间尿道HSV-2隐性排毒阳性率有显著性差异(P<0.05)。GH患者尿道HSV-2总阳性率为(22%),明显高于NGU组(9.8%)及正常组(3.3%);现症发作期GH患者尿道HSV-2阳性率(21.7%)与复发间歇期患者尿道HSV-2阳性率(23%)无显著差异(P>0.05)。每年复发6次以上的GH患者尿道HSV-2阳性率(27.4%)明显高于复发6次以下者(15%),且有显著性差异(P<0.05)。结论:GH患者发作期及间歇期尿道均有HSV-2隐性排毒。年复发频率在6次以上的GH患者HSV-2隐性排毒多于年复发频率6次以下者。病程3年以内者尿道隐性排毒较3年以上者高;Ct、Uu阴性的NGU患者部分可由HSV-2感染所致。