荧光多重PCR与血清型特异性抗体检测HSV感染的比较

目的：比较荧光多重PCR和血清型特异性抗体测定在生殖器疱疹临床诊断中的应用价值及评价各自的优缺点。方法：以细胞培养法作为“金标准”对照，分别用荧光多重PCR和血清型特异性抗体检测法对121例临床诊断为生殖器疱疹的标本进行检测。结果：以培养法作标准，并通过结果的差异性分析，荧光多重PCR的敏感性为100％，特异性为88．89％；血清型特异性抗体测定则分别为77．68％和77．78％，荧光多重PCR的敏感性和特异性均显高于血清型特异性抗体测（P＜0．05），但前不能检测出无皮损患HSV的DNA，而后可检测出无皮损患中的HSV抗体。结论：荧光多重PCR和血清型特异性抗体检测各有其自身的优缺点，单独用PCR和其它病毒分离的方法和单独使用血清特异性抗体检测的方法来诊断生殖器疱疹都是不完整的，均可造成漏诊。临床上将两种方法有机的结合起来应用能发挥各自的优势，取长补短，对早期、准确、快速地诊断生殖器疱疹及进行流行病学调查有着十分重要的意义和使用价值。     

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。     

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。     

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。     

Rapid detection of glycoprotein G gene for the diagnosis and typing of herpes simplex virus infection in genital herpes

OBJECTIVE: To develop a new, rapid, and convenient technique for the diagnosis and typing of herpes simplex virus (HSV) in genital herpes (GH). METHODS: Using samples from skin vesicle fluid and urogenital mucosal swabs of subjects with GH, conventional polymerase chain reaction (PCR) (directed to polymerase gene: PCRpG) were compared with a newly developed PCR (directed to HSV glycoprotein gene: PCRgG). Both PCR methods were compared with virus isolation culture (VI) with indirect immunofluorescent staining (IIF). RESULTS: 80 samples from 40 GH patients (25 males) were tested. Positive results were seen in 52.5% (42/80) using PCRgG compared with 40% (32/80) by VI. Most of PCRgG positive samples (95.1%) were caused by HSV-2 infection. In samples from healing lesions, HSV was detected more often by PCRgG, than by VI. The results of typing by PCRgG and IIF were highly consistent. CONCLUSION: PCRgG is more sensitive than VI and PCRgG in detecting HSV in urogenital samples from subjects with GH. PCRgG is a convenient technique for the rapid detection and typing of GH.     

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     

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     

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 of cutaneous herpes simplex infections using specific monoclonal antibodies

One hundred children with suspected herpes simplex virus (HSV) infection and 20 controls were studied to compare a rapid immunofluorescence (RIF) test for detection and typing of HSV from smears of lesions with standard viral culture. The RIF test was evaluated for ease of use and speed of diagnosis. RIF and/or culture were positive in 64% of patients. All infections diagnosed by RIF and culture were HSV type 1. In 92% of patients RIF and culture results were in concordance. In 57 cases, RIF and cultures were positive for HSV infection and in 35 cases RIF and cultures were negative for HSV infection. Three patients had inadequate samples for RIF and five children had positive RIF but were culture negative. All controls had negative results both by RIF test and culture. The RIF test demonstrated 100% sensitivity and 95% specificity. The RIF test was type specific, easy to perform and gave diagnosis of HSV infections within an hour of taking the clinical specimen. This study suggests the RIF test is as good, if not more sensitive, in the diagnosis of HSV infections as standard viral culture and has the advantage of speed of diagnosis.     

British Co-operative Clinical Group national survey on diagnostic issues surrounding genital herpes. MSSVD Special Interest Group on Genital Herpes and the British Co-operative Clinical Group

OBJECTIVES: To investigate the current use of diagnostic methods for genital herpes simplex virus (HSV) infection, to determine how information from these tests influences clinical practice, and to identify areas for future guideline development within genitourinary medicine (GUM) clinics in the United Kingdom. METHODS: National survey of 173 consultants in UK GUM clinics. RESULTS: Completed questionnaires were returned by 146 (84%) consultants. Cell culture was the first line diagnostic method for 133 (91%) respondents, the remaining 13 (9%) used antigen detection tests. Typing of isolates (HSV-1 or HSV-2) was available in their local laboratory to 109 (75%) clinicians; however, less than two thirds routinely pass this information on to their patients. Although 74 (51%) respondents had access to serological diagnosis, the majority of methods described were non-specific; only three (2%) had access to type specific tests. Only 81 (56%) respondents frequently (> 90% of the time) recommend notification of recent sexual partners of genital herpes patients. CONCLUSIONS: While access to culture based diagnosis is widespread, type specific serology has limited availability. Information on typing of isolates as HSV-1 or 2, although available in three quarters of centres, is underutilized in counselling patients. As HSV type influences both clinical and subclinical reactivation rates and may also affect probability of transmission, this is an important omission. Future guidelines need to address the optimal use of viral typing and new diagnostic tests to optimise health gain; there is also a need for evidence based recommendations about partner notification in genital herpes.     

生殖器部位皮损的单纯疱疹病毒检测及分型

目的 探讨生殖器疱疹部位皮损的不典型表现及其与单纯疱疹病毒型别的关系。方法 对外生殖器部位及其周围有硬结或疖肿、裂隙、毛囊炎等非水疱性皮肤黏膜损害的患者进行临床资料采集和分析,并对皮损标本进行单纯疱疹病毒的分离培养、PCR检测和病毒分型。结果 105例有外生殖器部位非水疱性皮损的患者入选本研究,在硬结(或疖肿)、裂隙、毛囊炎、类似擦破、单个溃疡、非特异性红斑和红肿渗液性包皮龟头炎皮损中,PCR检测HSV的阳性率分别33.3%(6/18)、20%(3/15)、37.5%(6/16)、28.6%(2/7)、33.3%(4/12)、20%(5/25)和50%(6/12),总的检出阳性率为30.5%(32/105)。分离培养法检测HSV的阳性率分别为22.2%(4/18)、13.3%(2/15)、25%(4/16)、14.3%(1/7)、33.3%(4/12)、8%(2/25)和41.7%(5/12),总的检出阳性率为21%(22/105)。两种方法检测HSV的总检出率差异无统计学意义(κ=0.095,P=0.114)。HSV-PCR分型结果与荧光单克隆抗体分型结果相符。在所有HSV阳性者中,HSV-1感染占9.4%(3/32),HSV-2感染占90.6%(29/32)。结论 生殖器HSV感染的皮肤黏膜损害多样,可为外生殖器部位的硬结(疖肿)、裂隙、毛囊炎、类似擦破、单个溃疡、非特异性红斑和红肿渗液性包皮龟头炎等不典型表现,而且主要由HSV-2感染引起。     

Detection of varicella zoster virus in genital specimens using a multiplex polymerase chain reaction

OBJECTIVE: To compare the relative proportions of varicella zoster virus (VZV) and herpes simplex viruses in specimens obtained from the genital lesions of adults presenting with presumed genital herpes infection. METHODS: Swabs of genital lesions from 6210 patients attending general practices, infectious diseases clinics within hospitals, or sexual health centres for treatment of their genital lesions were tested using polymerase chain reaction (PCR) technology. The multiplexed PCR was capable of detecting herpes simplex virus types 1 and 2 (HSV-1, HSV-2), VZV, and cytomegalovirus in a single sample. RESULTS: A total of 2225 patients had viruses detected by PCR. HSV-1 was detected in 36%, HSV-2 in 61%, and VZV in 2.9% of PCR positive samples. Of the 65 patients with VZV genital infection, many were thought to have HSV infection before laboratory testing. CONCLUSIONS: The finding of VZV in nearly 3% of virus positive genital specimens demonstrates that this virus needs to be considered as a differential diagnosis for genital herpetic lesions. Advice provided to patients with VZV genital infection regarding the source of infection, likelihood of recurrence, and potential for transmission of the virus will be different from that given to patients with HSV infection.     

First episodes of genital herpes in a Swedish STD population: a study of epidemiology and transmission by the use of herpes simplex virus (HSV) typing and specific serology

OBJECTIVES: To determine the proportion of herpes simplex virus type 1 (HSV-1) and HSV type 2 (HSV-2) in first episodes of genital herpes. To evaluate the use of HSV specific serology for classifying first episodes of genital herpes and for defining HSV serostatus in the patients' sexual partners. METHODS: 108 consecutive patients with first episodes of genital herpes seen at three STD clinics in Sweden from 1995 to 1999 were included in the study. HSV culture and typing were performed and serum was tested for antibodies against a type common HSV antigen and a type specific HSV-2 antigen, glycoprotein G2 (gG2). A structured interview including questions about sexual behaviour and sexual partners was taken. "Steady" partners were offered a blood test for HSV serology and counselling. RESULTS: Of 108 patients, 11 had a negative HSV culture. Of the 97 who were HSV culture positive, 44% (43/97) were typed as HSV-1 and 56% (54/97) as HSV-2. For 86 of these 97 patients, HSV serology from the initial visit was available. Of 52 primary infections, thus initially seronegative, 64% were HSV-1 infections and of 19 female primary infections 16 (84%) were HSV-1. In 17% the first episode of genital herpes corresponded to the first clinical recurrence of an infection acquired earlier in life. There was a significant correlation between having orogenital sex and being infected with HSV-1 and also a history of labial herpes in the partner. Only 20% of partners of patients with an HSV-2 infection had a history of genital herpes. CONCLUSIONS: Almost half of first episodes of genital herpes are caused by HSV-1. In young women with a primary genital infection, HSV-1 is much more frequent than HSV-2. Besides HSV typing, we found specific HSV serology of value for classifying first episodes and for diagnosing a subclinical HSV-2 infection in partners. Anamnestic data supported the suggestion that the orogenital route of transmission was common in genital HSV-1 infections.     

Rapid amplified enzyme linked immunosorbent assay evaluated for detecting herpes simplex virus.

A new amplified enzyme linked immunosorbent assay (amplified ELISA) kit for detecting herpes simplex virus (HSV) antigen was evaluated. Duplicate swabs were taken from 180 patients with clinically suspected herpes lesions. Tests were performed on a direct swab extract and viral transport medium containing a swab. Of the 93 culture positive specimens, 78 of the extracted samples (sensitivity 83.9%) and 72 of the swabs in transport medium (sensitivity 77.4%) were positive by amplified ELISA. A higher sensitivity (49/54, 90.7%) was obtained when the extracted swab was taken first. In early lesions the sensitivity was 93.8% but in late lesions it was 73.3%. This ELISA therefore offers an alternative to culture for early lesions, but culture is the method of choice for differential diagnosis of genital ulceration. As the specificity was 94.3%, this test is acceptable for testing populations with a high prevalence of HSV infection, but culture should be used for screening populations in which the disease is rare.     

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.     

One-day regimen of valacyclovir for treatment of recurrent genital herpes simplex virus 2 infection

OBJECTIVES: To evaluate the efficacy of a 1-day course of valacyclovir in reducing the duration and severity of genital herpes recurrences and to measure the frequency of viral shedding episodes subsequent to antiviral therapy. STUDY DESIGN: In an open-label pilot study, patients with recurrent genital herpes simplex virus 2 (HSV-2) infection were given a 1-day course of valacyclovir (2000 mg given by mouth twice daily) to be taken at the first sign of recurrence or prodrome. Participants maintained diaries of signs and symptoms and collected genital swabs for viral culture while lesions persisted and HSV DNA PCR for 14 days after initiating treatment. RESULTS: Ninety (78%; 41 men, 49 women) of the 115 enrolled persons experienced either a lesional recurrence or prodrome. Seventy-seven (86%) participants developed lesions; 4 (5%) participants experienced a second lesional recurrence during the 14-day study period. The median lesion duration was 5 days, episode duration was 5 days, and pain duration was 3 days. Viral shedding was detected in 60 persons by PCR and 31 persons by culture. Shedding detected by culture lasted for a median of 2 days, and shedding detected by PCR lasted for a median of 3 days. Of 60 participants with viral shedding, 14 (23%) had an additional shedding episode after their initial lesion healed, lasting for a median of 2 days. CONCLUSIONS: A 1-day course of valacyclovir may be a convenient treatment for recurrent genital herpes and comparative trials are warranted.     

Survey of diagnostic services for genital herpes in fourteen countries in Eastern Europe

This paper reports survey-based data on the diagnosis and management of genital herpes simplex virus (HSV) infection in 14 countries of the Eastern European Network for Sexual and Reproductive Health (EE SRH). Only 43% of the countries could provide the number of genital HSV cases recorded at national level. Eighty-six percent of countries employed syndromic management in cases of genital ulcer disease. Most countries performed type-specific and/or non-type-specific enzyme immunoassays to detect HSV antibodies. Non-type-specific serology for diagnostic purposes should be actively discouraged. Direct detection methods for HSV, such as PCR, antigen detection and culture, are available in the region, but their usage was extremely low. Their use in Eastern European countries should be actively promoted. The availability of laboratory services must be improved, and countries in the region should implement consensus recommendations for the laboratory diagnosis of genital HSV infections in order to improve clinical practice.     

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     

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.