Time-resolved fluorometry PCR assay for rapid detection of herpes simplex virus in cerebrospinal fluid

We have introduced a time-resolved fluorometry (TRF)-based microwell hybridization assay for PCR products in detection of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive detection technique which involves the use of lanthanide chelates as fluorescent labels. We used PCR primers from the glycoprotein D genes of HSV type 1 (HSV-1) and HSV-2. The biotinylated PCR products were collected on streptavidin-coated microtitration wells and hybridized with short oligonucleotide probes, europium labeled for HSV-1 and samarium labeled for HSV-2. The TRF results were obtained as counts per second and as signal-to-noise (S/N) ratios. The sensitivity of the assay was 0.1 infectious units (PFU) of HSV in CSF specimens, and the S/N values increased with the virus amount, up to 68.5 for 10(3) PFU of HSV-1 and to 58.5 for 10(3) PFU of HSV-2, allowing semiquantitation of HSV in CSF. The primers and probes recognized all the studied 48 HSV wild-type samples, with S/N ratios of 12.4 to 190 (HSV-1) and 5.1 to 248 (HSV-2). We tested CSF specimens, 100 for each HSV type, which were HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 or -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-negative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-negative CSF it was 1.03 (SD = 0.098). The HSV-1 blot-positive CSF yielded S/N ratios of 3.6 to 85.9, and the HSV-2 blot-positive CSF yielded ratios from 1.9 to 13. Using the mean S/N ratio for negative CSF specimens + 3 SD as the cutoff yielded all the previously HSV-positive specimens as TRF positive. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive method, improves interpretation of PCR results, and is well suited for automation.     

Rapid detection of herpes simplex virus DNA in cerebrospinal fluid: comparison between loop-mediated isothermal amplification and real-time PCR

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. To evaluate the usefulness of LAMP for diagnosing central nervous system infection with herpes simplex virus (HSV), we compared the LAMP method with real-time PCR, using samples that were previously tested by nested PCR. We examined 69 cerebrospinal fluid (CSF) samples from patients suspected of having HSV infection of the central nervous system. The results of the real-time PCR analysis and nested PCR assay were in complete accord. When nested PCR was regarded as standard, the sensitivity of LAMP was 81%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 90%. Although further improvement is necessary for the wide spread use, the LAMP method might be applicable to diagnosis of HSV infection of the central nervous system.     

Screening for possible failure of herpes simplex virus PCR in cerebrospinal fluid for the diagnosis of herpes simplex encephalitis

The objectives of this study were to evaluate the reliability of herpes simplex virus (HSV) PCR testing in cerebrospinal fluid (CSF) for the detection of herpes simplex encephalitis. This was done by examining retrospectively the clinical follow-up of a large group of patients tested routinely by HSV-PCR. In addition, an attempt was made to assess the incidence of herpes simplex encephalitis in a central European population. CSF samples from 1,427 patients from all Vienna hospitals were submitted for HSV-PCR testing during a period of 4 years and 8 months. Herpes simplex encephalitis was detected by PCR in 12 cases and by serological methods in one additional patient. Retrospective analysis of the course of disease, which was possible in 799 PCR-negative patients, led to the identification of three additional cases in which herpes simplex encephalitis appears to have occurred despite negative PCR results. Failure of the PCR in these patients is most likely due to the time of obtaining CSF during the course of disease. A high specificity of the assay was demonstrated by the lack of false positive results in any of the 708 cases in which other causes for the neurological symptoms had been identified in the follow-up. The incidence of herpes simplex encephalitis in the population of Vienna was between 1 case/469,000-577,000 individuals/year. The highest annual incidence was detected in the age group between 3 months and 3 years, which, however, could not be confirmed statistically.     

Rapid susceptibility testing for herpes simplex virus type 1 using real-time PCR

BackgroundSusceptibility testing of herpes simplex virus type 1 (HSV-1) is traditionally performed by a plaque reduction assay (PRA), but this is labor intensive, time consuming and has a manual read out.ObjectivesThe goal of this study was to develop an internally controlled real time PCR-based phenotypical susceptibility test for HSV-1 that is suitable for use in a clinical diagnostic setting.Study designA DNA reduction assay (DRA) was developed and validated on a test panel of 26 well-characterized isolates of varying susceptibility to aciclovir or foscarnet, including low-level resistant isolates. The DRA consisted of pre-culture of a clinical sample for 48 h and subsequent culture in the presence of antivirals for 24 h. Viral DNA concentration in the culture lysates was measured by an internally controlled quantitative real-time HSV-1 PCR and corrected for cell count and lysis by beta-globin PCR. DRA results were compared to results from PRA and sequence analysis.ResultsDRA results were in accordance with PRA results for both aciclovir and foscarnet susceptibility and appeared to have good discriminative value for low-level resistance due to UL30 gene mutations. Although the direct application of DRA in clinical samples appeared not possible, short pre-culture of 48 h was sufficient and ensured results within a clinically relevant time frame of 5 days.ConclusionsDRA is an accurate, rapid and easy to perform phenotypical susceptibility test for HSV-1.     

Rapid detection of herpes simplex virus and varicella-zoster virus infections by real-time PCR

The herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV) can cause life-threatening infections of the central nervous system and lead to severe infections in immunocompromised subjects and newborns. In these cases, rapid diagnosis is crucial. We developed three different real-time PCR assays based on TaqMan chemistry for the LightCycler instrument to detect HSV-1, HSV-2, and VZV. When the TaqMan assays were compared to our in-house nested PCR assays, the test systems had equal sensitivities of 相似文献   

Increased cell-mediated immune responses in patients with recurrent herpes simplex virus type 2 meningitis

The clinical picture of herpes simplex virus type 2 (HSV-2) infection includes genital blisters and less frequently meningitis, and some individuals suffer from recurrent episodes of these manifestations. We hypothesized that adaptive and/or innate immune functional deficiencies may be a major contributing factor in susceptibility to recurrent HSV-2 meningitis. Ten patients with recurrent HSV-2 meningitis were studied during clinical remission. For comparison, 10 patients with recurrent genital HSV infections as well as 21 HSV-seropositive and 19 HSV-seronegative healthy blood donors were included. HSV-specific T cell blasting and cytokine secretion were evaluated in whole blood cultures. HSV-2-induced NK cell gamma interferon production, dendritic cell Toll-like receptor (TLR) expression, and TLR agonist-induced alpha interferon secretion were analyzed. Patients with recurrent HSV-2 meningitis had elevated T cell blasting and Th1 and Th2 cytokine production in response to HSV antigens compared to those of patients with recurrent genital infections. A somewhat increased NK cell response, increased dendritic cell expression of TLR3 and -9, and increased TLR-induced alpha interferon responses were also noted. Contrary to our expectation, recurrent HSV-2 meningitis patients have increased HSV-specific adaptive and innate immune responses, raising the possibility of immune-mediated pathology in the development of recurrent HSV2 meningitis.     

Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay

Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the "gold standard." From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain.     

Simultaneous detection of herpes simplex virus types 1 and 2 by real-time PCR and Pyrosequencing.

BACKGROUND: Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions. OBJECTIVE: To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype. STUDY DESIGN: ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5' biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced. RESULTS: The quantitative range of the assay extended from 10(8) through 10(0) copies of each virus (r(2) > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2. CONCLUSIONS: This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.     

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques.     

Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation.

BACKGROUND: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. OBJECTIVE: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the 'in-house' nested PCR and virus isolation. STUDY DESIGN: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. RESULTS: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. CONCLUSION: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions.     

Laboratory diagnosis of central nervous system infections with herpes simplex virus by PCR performed with cerebrospinal fluid specimens.

Until recently, the laboratory diagnosis of central nervous system (CNS) infections with herpes simplex virus (HSV) has been limited by poor sensitivity and/or specificity. We assessed the diagnostic utility of PCR for detection of HSV in over 2,100 specimens referred to the Mayo Clinic from August 1993 to May 1996. DNA extracted from cerebrospinal fluid (CSF) samples with IsoQuick was amplified by PCR with oligonucleotide primers directed to the DNA polymerase gene of HSV, yielding a 290-bp amplicon. HSV DNA was detected in 150 (135 by gel electrophoresis, 15 by Southern blotting only) of 2,106 (7.1%) specimens. PCR-positive CNS disease occurred in patients ranging in age from 13 days to 89 years; 59% of the cases occurred in patients between the ages of 30 and 69, and 21 (14%) of the patients were infants. Genotype analysis was not routinely performed; however, amplification of a 335-bp product within the thymidine kinase gene of HSV revealed 13 positions within a span of 80 nucleotides that accurately identified the two serotypes of the virus according to 14 reference strains. We conclude that PCR detection of HSV DNA in CSF specimens should be considered an emerging "gold standard" for the laboratory diagnosis of CNS infections with this virus.     

Monitoring of herpes simplex virus DNA types 1 and 2 viral load in cerebrospinal fluid by real‐time PCR in patients with herpes simplex encephalitis

A quantitative polymerase chain reaction (PCR) assay was evaluated retrospectively on 92 cerebrospinal fluid (CSF) samples from 29 patients with herpes simplex virus (HSV) encephalitis with the aim to study if the concentration of HSV genomes can be used as a prognostic marker and for monitoring of antiviral therapy. The results were compared to those obtained previously by nested PCR, and the numbers of HSV genomes/ml were evaluated in correlation to patient outcome and treatment. The aims were to compare the sensitivity of a conventional nested PCR to a quantitative PCR, to investigate the range of HSV genome concentration in initial samples and to evaluate possible relationships between the HSV DNA concentrations in CSF, neopterin levels, and outcome of disease. The 29 initial samples contained between 2 × 10² and 42 × 10⁶ HSV genomes/ml. There was no apparent correlation between the amount of HSV DNA in the initial samples and income status, initial neopterin levels, or prognosis. The number of HSV genomes/ml declined after treatment in all patients, but HSV DNA was still detectable after day 20 in 3 out of 16 patients. A long duration of genome detectability was found to correlate with poor outcome. There was no difference in sensitivity between the nested PCR and the quantitative PCR. While the quantitative PCR is more rational than a nested PCR, the quantitation of HSV genomes does not seem very useful as a prognostic marker in HSV encephalitis. J. Med. Virol. 81:1432–1437, 2009. © 2009 Wiley‐Liss, Inc.     

Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal

We have evaluated a real-time PCR procedure based on the LightCycler technology for rapid detection of herpes simplex virus (HSV) in genital lesions. Two sets of primers, corresponding to the thymidine kinase and DNA polymerase regions, were used for the amplification reactions in separate capillaries containing the SYBR Green I dye as detection signal. In 28 of 118 samples (24%), HSV was isolated by conventional cell culture. All cell culture-positive samples were also positive by real-time PCR. Six additional cell culture-negative samples were positive by PCR with both sets of primers. Total processing time was less than 3 h. Real-time PCR using SYBR Green I as detection signal is a sensitive procedure for the rapid diagnosis of HSV in genital lesions.     

Detection of herpes viruses in clinical samples using real-time PCR

Herpes viruses including cytomegalovirus, varicella zoster and herpes simplex are an important cause of morbidity and mortality, especially in immunocompromised patients. Real-time PCR assays were developed with the aim of introducing a rapid and sensitive test to replace culture, and as a surveillance system for high-risk patients. The assays were optimised using cell culture derived material, and the sensitivity ascertained using cloned product before applying to extracted and non-extracted clinical samples. The sensitivity was between 1--100 virus copies with increased sensitivity to detect less than 10 copies possible when an initial round of amplification was carried out using external primers. Results were available within four hours of receipt compared with a median of 4.4 days for culture and immunofluorescence. Real-time PCR was found to be a sensitive and rapid method of detecting these viruses and will be a valuable tool for the surveillance of immunosuppressed patients.     

Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique.

A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutoff was determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.     

ELISA for the detection of herpes simplex virus antigens in the cerebrospinal fluid of patients with encephalitis

An inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of herpes simplex virus antigens in cerebrospinal fluid (CSF) has been developed. A Triton X-100 extract of herpes simplex virus type 1 (HSV-1) infected HEp-2 cells was used to coat wells of polyvinyl chloride plates. Rabbit anti-HSV-1 globulin served as the reference antibody and the CSF specimens were tested at a final dilution of 1:4. Positive results were obtained in CSF specimens from 11/18 (61%) neonates with HSV infection, 15/23 (65%) older individuals with HSV culture positive brain biopsies, and in 4/29 (14%) patients with culture negative brain biopsies. The assay was negative with CSF from 14 infants without HSV infections, from 30 patients with bacterial meningitis and 10 with cryptococcal meningitis. The test was positive in 10/21 patients within 10 days of onset, 11/14 within 11-20 days, and in 5/6 more than 20 days after onset of the herpetic infection. The overall sensitivity of the assay was 63% and the specificity was 95%.