Use of the nested polymerase chain reaction in the differential diagnosis of human herpes simplex virus

Herpes is one of the most widespread human viral pathologies. The article depicts a special modification of polymerized chain reaction--(PCR)--(referred to as "nested"), which has a higher sensitivity, specificity and reliability as compared to the ordinary PCR, and which is designed to detect the herpes viruses. The method was initially tested at purified preparation of viral DNA, and later--at clinical materials obtained from patients and healthy donors. Secretions from the urogenital tract (smears), scrapes from the urogenital tracts and urinal cellular samples were examined in patients. Herpes simplex was detected in all cases. As for the healthy people, the identical examinations produced in them mainly the negative findings. Therefore, the nested PCR is a simple, sensitive and effective instrument in the diagnostics and prevention of herpes; it can be recommended for a comprehensive usage in medical practice.     

Detection of herpes simplex virus type 1 in addition to Epstein-Bar virus in tonsils using a new multiplex polymerase chain reaction assay

HSV-1, HSV-2, CMV, EBV, which are the members of the herpes virus family colonize and establish latent infection in human. Although EBV is a well known virus most involved in recurrent bouts of acute tonsillitis, the role and possibility of HSV-1, HSV-2, and CMV for establishing infection in tonsils are not clear. The purpose of this study is to verify whether the tonsils might harbor the HSV-1, HSV-2, and CMV, in addition to EBV, in chronically hyperplastic nasopharyngeal lymphoid tissue. To accomplish the purpose, we developed a new Multiplex Polymerase Chain Reaction (M-PCR) assay using a single consensus forward primer and virus specific reverse primers for DNA polymerase gene of HSV-1, and 2, EBV, and CMV, and investigated its efficiency for detecting HSV1, HSV2, CMV, and EBV. The sample of 52 patients underwent tonsillectomy or adenectomy because of chronic lymphoid hyperplasia without any evidence of acute infections and were investigated for presence of HSV-1, HSV-2, CMV, and EBV. Of the 54 samples, 11 (20.4%) of them were positive for EBV, 4 of them (7.4%) were positive for HSV-1, and none of the samples were positive for HSV-2 and CMV. To the best of our knowledge, this is the first report that tonsils may be the reservoir for HSV-1 in addition to EBV, and HSV-1 may have a role in recurrent tonsillitis and systemic diseases. The MC-PCR assay presented in this study can provide a rapid, sensitive, and economical method for detection of HSV-1, HSV-2, EBV, and CMV in a single PCR tube.     

Rapid electrochemiluminescence assays of polymerase chain reaction products.

We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.     

A prospective study of the polymerase chain reaction for detection of herpes simplex virus in cerebrospinal fluid submitted to the clinical virology laboratory.

Polymerase chain reaction (PCR) was prospectively performed with cerebrospinal fluid (CSF) from 51 patients whose CSF was available for analysis and was submitted for viral culture and/or herpes simplex virus (HSV) serology and 20 patients whose CSF was submitted exclusively to the Clinical Biochemistry Laboratory. Primers were used that flanked a 92 bp segment of the HSV DNA polymerase gene (35 cycles). Amplified products were electrophoresed on agarose gel, blotted onto nylon membrane, and probed with a 32P-labelled sequence internal to the primers. For nested PCR, 1 microliter of PCR product was amplified for an additional 35 cycles before electrophoresis and Southern blot analysis. Review of the clinical records revealed that 15 patients had central nervous system (CNS) infections. Specific HSV DNA sequences were detected in CSF specimens of three of the individuals [PCR(2), nested PCR(1)]. Two of these patients had disseminated HSV infection including encephalitis and one patient had aseptic meningitis. The diagnoses of the 12 patients with CNS infection who did not have HSV DNA detected in CSF included encephalitis [varicella-zoster virus (1), cytomegalovirus (1), Mycoplasma pneumoniae (1)], meningitis [Neisseria meningitidis (1), Coccidioides immitis (1), Enterovirus (1), aseptic meningitis (1)], varicella-zoster radiculitis (2), human immunodeficiency virus dementia (2), and transverse myelitis due to Epstein-Barr virus (1). Importantly, HSV DNA was also not detected in the CSF of the 36 patients who did not have CNS infection and 20 samples submitted exclusively to the Clinical Biochemistry Laboratory. Our findings demonstrate the utility of PCR as a rapid, non-invasive method for the routine laboratory diagnosis of CNS infection due to HSV.     

Real-time polymerase chain reaction detection of herpes simplex virus in cerebrospinal fluid and cost savings from earlier hospital discharge

Neonatal herpes simplex virus (HSV) can be a devastating illness and may be difficult to diagnose in those cases without a typical skin rash. As a result, physicians often rely on HSV polymerase chain reaction of cerebrospinal fluid to rule out HSV encephalitis. We developed a real-time polymerase chain reaction assay for HSV using the SmartCycler II (Cepheid, Sunnyvale, CA). End point dilution studies showed sensitivity comparable to that of two national reference laboratories that use LightCycler. In-house turnaround time was approximately 1.5 days versus approximately 5.2 days for sending the test to a reference laboratory. We hypothesized that the rapid availability of a negative test result would allow physicians to discharge appropriate patients earlier. Six months after implementation, clinical case analysis identified 12 pediatric patients who were discharged earlier based on more rapid test results, with a projected savings of approximately 55.2 hospital days throughout the first year. Actual length of stay for patients tested in-house was significantly less than that of historical controls and was projected to save approximately 70.2 hospital days in the first year. Including projected annual laboratory cost/test savings of approximately $11,000, a total savings of $38,000 to $43,000 was estimated for the first year of implementation, more than offsetting startup instrument and development cost.     

非淋菌性尿道炎患者单纯疱疹病毒感染的研究

目的　研究非淋菌性尿道炎患者单纯疱疹病毒的感染情况。方法　采用聚合酶链反应法对NGU患者组15 6例及对照组 6 0例进行HSV检测。结果　NGU患者组HSV阳性率为 2 1 79% ,对照组阳性率为 5 0 0 % ,患者组与对照组比较差异有显著性意义 (χ2 =8.6 1,P >0 0 0 5 )。结论　HSV是NGU的重要病原体之一 ,PCR作为HSV感染的实验诊断技术具有很好的应用价值     

Antibodies against herpes simplex virus in a group of healthy humans without reactions to herpes simplex virus antigens in blasttransformation assays

22 healthy individuals with different reactions in humoral and cell-mediated immunity to herpes simplex virus (HSV), as found in complement-fixation, neutralization, and blasttransformation assays have been further investigated using antibody-dependent cell-mediated immunity (ADCC) as a serological test. Sera from all individuals were absorbed on HSV-infected, varicella zoster virus (VZV)-infected or uninfected cell monolayers, before they were used in ADCC with HSV-infected cells, as target. The results with absorbed sera were compared with the results from unabsorbed sera. Fresh uninactivated sera have also been used in some of the investigations. By these experiments a group of individuals was found, who were HSV-positive in ADCC, but negative in all other, above-mentioned tests. Because of these reactions they are proposed to be HSV-positives without a latent infection.     

Retention of nonlinear viral DNA during herpes simplex virus latency in vitro.

We investigated the state of the herpes simplex virus type 2 (HSV-2) genome during latency in vitro. Latent DNA was present in the cell nucleus in a nonlinear configuration. The joint fragment was represented at approximately double the molar concentration of fragments from unique regions, indicating that genome termini had fused. The HSV-2 genome copy number was estimated to be between 1.5 and 8, with a mean value of 4.5 per latently infected cell. Nonlinear HSV DNA can therefore exist during latency both in vivo and in vitro.     

Appropriate use of polymerase chain reaction for detection of herpes simplex virus 2 in cerebrospinal fluid of patients at an inner-city hospital

Polymerase chain reaction (PCR) tests that detect herpes simplex virus (HSV) DNA in cerebrospinal fluid (CSF) are increasingly used to diagnose central nervous system (CNS) infections caused by HSV. To determine proper utilization of this test at an inner-city hospital, we performed a case-control study of adult patients, with HSV detected in CSF by PCR. Retrospective review of characteristics of adult patients hospitalized between 1997 and 2000 with CSF positive for HSV was done and compared to control patients with suspected CNS infection and negative CSF PCR. CSF from 1174 patients was tested; 20 (1.7%) had HSV DNA detected, 19/20 were HSV-2 and 1 was HSV-1. The HSV-2 cases were females (74%), with a median age of 41 years, of African-American ethnicity (100%). Of the cases, 90% had acute aseptic meningitis versus 13% controls (P < .001). Recurrent meningitis occurred in 42% cases and 3% controls (P < .001). CSF parameters significantly associated with HSV-2 positivity was lymphocytic pleocytosis (median leukocyte, 475 cell/mm3, 90% lymphocytes) (P < .001). In conclusion, HSV-1 was rarely detected in CSF of patients with suspected CNS infection. HSV-2 is more frequent, predominantly in young African-American women with lymphocytic aseptic meningitis, and is often recurrent. PCR testing for HSV-2 in CSF at inner-city hospitals can be greatly reduced by the application of these parameters.     

Concurrent detection of herpes simplex and varicella-zoster viruses by polymerase chain reaction from the same anatomic location

Herpes simplex virus (HSV) and varicella-zoster virus (VZV) may cause latent infection of the same peripheral nerve ganglia. However, there are no large studies addressing the frequency of concurrent HSV/VZV PCR positivity from the same anatomic location. In an eight-year retrospective study, we observed 1.3% dual positivity from dermal, genital, and oral mucosal sources.     

Detection of herpes simplex virus type 2 Bgl II N fragment in paraffin-embedded cervical tissue sections using nested polymerase chain reaction

To study the presence of transforming sequence Bgl II N of HSV-2 in cervical cancer tissues, we developed the nested polymerase chain reaction (PCR) for detecting such a sequence in paraffin-embedded cervical tissue sections. Samples derived from 46 patients with premalignant and malignant lesions were tested. The sequence was found in 20-25% of total tested but not observed in any of the normal healthy controls. This study also indicates that for the detection of HSV-2 Bgl II N sequence in cervical tissue, the nested PCR may be more reliable than the in situ hybridization method.     

A novel protein tag from herpes simplex virus type 1 DNA polymerase.

An epitope (HPOL) derived from the so-called thumb region of the herpes simplex virus type 1 DNA polymerase in combination with a monoclonal antibody (MAb 1051c) was tested for protein tagging. Using a conventional expression vector, a DNA cassette encoding the HPOL epitope was fused to the C-terminus of the dihydrofolate reductase (DHFR) gene such that the recombinant DHFR contained both a N-terminal HIS-tag and a C-terminal HPOL tag. Expression of recombinant DHFR in Escherichia coli cells was compared by Western blot analysis using either mouse RGS.HIS antibody or MAb 1051c. Immunostaining revealed that both antibodies reacted specifically with DHFR, but the detection sensitivity achieved with MAb 1051c was about 15-fold greater using a standard staining protocol. An HPOL antibody column was successfully applied for affinity purification of DHFR, demonstrating the usefulness of the HPOL epitope/MAb 1051c system for protein tagging, expression monitoring and purification of HPOL-tagged recombinant proteins.     

Genital herpes simplex virus infections.

In recent years, a great increase in interest in genital herpes has been stimulated partly by the rising prevalence of this disease and partly by observations suggesting that genital herpes is a cause of cervical cancer. The clinical pictures produced by genital herpes simplex virus infections are similar in men and women. In contrast to recurrent attacks, initial episodes of infection are generally more extensive, last longer, and are more often associated with regional lymphadenopathy and systemic symptoms. Genital herpes in pregnancy may pose a serious threat to the newborn infant. Although the data suggesting genital herpes simplex virus infection is a cause of cervical cancer are quite extensive, the evidence is largely circumstantial. In spite of these more serious aspects of genital herpes simplex virus infection, episodes of genital herpes are almost always self-limited and benign. Frequent recurrences pose the major therapeutic and management problem. At present, there is no satisfactory treatment for recurrent genital herpes simplex virus in fection. Many of the suggested therapies, although some sound very promising, are potentially dangerous and should be used only under carefully controlled conditions.     

Cutaneous herpes simplex virus infections.

Affecting millions of Americans each year, herpes simplex virus infections are among the most common human viral infections. Many clinical forms exist, depending on the site of infection and the patient's age and immune status. Clinical evaluation and laboratory studies help establish the diagnosis. Acyclovir is the drug most often used to treat herpes simplex virus infections, although newer agents, such as phosphonoformate trisodium, may be required for acyclovir-resistant infections.     

Development and evaluation of dual-target real-time polymerase chain reaction assays to detect Bordetella spp

Novel, highly specific, and sensitive real-time polymerase chain reaction (PCR) assays using 2 targets, insertion sequence (IS481) and pertussis toxin subunit 1 (ptxS1), were developed to detect Bordetella pertussis and to differentiate between relevant Bordetella spp. Sixty-four non-Bordetella isolates were negative by both assays, demonstrating the specificity of the assays. B. pertussis, Bordetella parapertussis, and Bordetella holmesii isolates were specifically identified using the assays. The lower limit of detection was less than 10 genomic equivalents per reaction for the IS481 and ptxS1 assays. These assays were evaluated using 145 human clinical specimens obtained during cough-illness outbreak investigations, and PCR results were compared with Bordetella spp. culture results. Twenty-seven (18.6%) specimens had late positive cycle threshold (Ct) values (35 相似文献   

Detection of vaccinia virus,herpes simplex virus,varicella-zoster virus,and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving: implications for biosafety of bioterrorism agents

OBJECTIVE: To determine whether autoclaving suspensions of vaccinia virus, herpes simplex virus (HSV), varicella-zoster virus (VZV), and Bacillus anthracis inactivate infectivity of these agents but allow detection of target DNA by LightCycler polymerase chain reaction (PCR). MATERIAL AND METHODS: Swabs were inserted into tubes containing serial 10-fold dilutions (10(-1) to 10(-5); 500 microL; 6 samples per dilution) of vaccinia virus, HSV, VZV, or a single suspension of 10(8) colony-forming units of B anthracis (2 samples). One half of the samples were autoclaved, and the remainder were not. An aliquot of each not autoclaved sample served as a control for infectivity. RESULTS: Autoclaving swabs saturated with suspensions of vaccinia virus, HSV, or VZV eliminated the infectivity of these agents; however, DNA was detectable in most autoclaved samples in dilutions of 10(-1) to 10(-4) by LightCycler PCR. All not autoclaved specimens were detected by culture (infectivity) except for VZV and, in most dilutions of 10(-1) to 10(-3), by assay of target DNA by LightCycler PCR. Similarly positive results were obtained for PCR assessment of sporulated B anthracis. CONCLUSIONS: Standard autoclaving procedures eliminated the infectivity of viruses (and B anthracis), but target DNA was often retained for detection by LightCycler PCR. Current recommendations indicate that the laboratory diagnosis of smallpox virus infection be performed only within Biosafety Level 4 facilities. We suggest that, in addition to the requirement for immediate coordination with public health officials, the federal government consider expanding the existing guidelines for processing these specimens to encourage immediate collection, autoclaving, and testing by LightCycler PCR to differentiate smallpox virus from other dermal pathogens such as HSV and VZV by specific qualified laboratories.     

Development of an internally controlled, homogeneous polymerase chain reaction assay for the simultaneous detection and discrimination of herpes simplex virus types 1 and 2 and varicella-zoster virus

Homogeneous polymerase chain reaction (PCR) technology is being used increasingly in the diagnosis of infectious disease. The sensitivity and specificity of PCR is being coupled to the ease-of-use and multiplexing capacity of homogeneous methodologies to provide rapid and accurate differential diagnoses. This technology is applicable to the diagnosis of infections with the human herpes viruses, herpes simplex virus 1 (HSV 1), HSV 2 and varicella-zoster virus (VZV). Our aim was to develop and evaluate a homogeneous PCR assay which combines the following features: the assay can detect and distinguish HSV types 1 and 2 and VZV, can be performed on untreated clinical samples, contains internal control reagents to monitor for inhibitors in the sample and allows automatic assignment of viral genotypes. Primers and probes specific for HSV and VZV genes were combined and optimized in a multiplex PCR. An internal control was designed which allowed use of the VZV primers and a human factor V gene DNA template. The assay was evaluated on an initial cohort of 66 clinical swab samples, with results determined by visual inspection of melt curves. Parameters obtained from this study were used to assign genotypes automatically to a second group of 85 clinical swab samples. Optimization of reagents produced melt curve peaks of sufficient height and symmetry for automatic genotype assignment. In the initial cohort of 66 samples, 63 returned concordant results, one sample produced an aberrant peak due to sequence variation and the remaining two samples were positive on re-test. Automatic genotype assignment of the second group of 85 samples resulted in correct identification of 79 samples, with two further aberrant peaks, and two samples positive on retest. The development of this assay should facilitate the rapid detection of herpes viruses from clinical swab samples.