Detection Of Herpes Simplex Virus Research Articles

A flexible and low-cost polypropylene pouch for naked-eye detection of herpes simplex viruses.

Effective viral detection is a key goal in the development of point of care (POC) diagnostic devices. Loop-mediated isothermal amplification (LAMP) could potentially be a valuable tool for rapid viral detection and diagnosis in commercial and hospital laboratories and resource limited settings. Here, we present a novel polypropylene pouch (PP) for detection of HSV-1 and HSV-2. With this plastic pouch we could detect up to 6.08 × 10(1) copies per μL of HSV-1 DNA and 0.598 copies per μL of HSV-2 DNA within 45 minutes. Since LAMP itself is less sensitive to inhibitory substances present in the real sample, we could also detect viral DNA without the need for viral DNA extraction and purification. The result from LAMP could be evaluated by naked eye due to the addition of hydroxy naphthol blue (HNB) dye in the reaction mixture. Since this proposed device is easy to handle, portable, user friendly and low cost, it offers a tremendous potential to be a perfect candidate for POC diagnostic device for use in resource limited settings.

Comparison of the Simplexa HSV1 & 2 Direct kit and laboratory-developed real-time PCR assays for herpes simplex virus detection

BackgroundRapid detection and differentiation of herpes simplex viruses (HSV) is important for patient management and treatment, especially in HSV meningoencephalitis. ObjectivesResults of Simplexa HSV1 & 2 Direct kit (Focus Diagnostics), an FDA-cleared sample-to-result method providing results in ∼75min, were compared to those of laboratory-developed real-time PCR assays (LDT) for detection of HSV1 and HSV2. Study designSamples tested included 168 cerebral spinal fluid (CSF) collected prospectively and 150 tested retrospectively: 81 from clinical testing and 69 from subjects in a neonatal herpes study; and 53 plasma and sera. Each sample was tested by both methods on the same day. ResultsThree of 318 CSF had invalid Simplexa Direct results and negative LDT results. Three neonatal samples with low HSV viral loads by LDT could not be typed; two were HSV2 positive and one was negative by Simplexa Direct. Of 312 CSF with valid, type-specific results, HSV1 was detected in 16 by LDT and in 17 by Simplexa Direct; HSV2 was detected in 48 by LDT and in 49 by Simplexa Direct. Concordance rates were 98.4% (κ 0.84) and 97.1% (κ 0.89) for HSV1 and HSV2, respectively. Positive percent agreements were 87.5% for HSV1 and 91.7% for HSV2. Two and four CSF were positive only by LDT and three and five were positive only by Simplexa Direct for HSV1 and HSV2, respectively. ConclusionsSimplexa HSV1 & 2 assay performed well compared to an established LDT. The faster turn-around-time compared to LDT will allow for more rapid antiviral treatment and better patient management.

Hormone Level in Uterine Carcinoma Tissue Associated with Different Viral Infections

ABSTRACT Aim: Unpredictability of tumor biological behavior may be determined by the involvement of different infectious agents in carcinogenesis. Malignant tumor's independent synthesis of hormones contributes to autonomy of growth and absence of central regulation. The question of the influence of infectious agents on hormonal status of malignant uterine tumors still remains open. Methods: Levels of estrone (Е1), estradiol (Е2), testosterone (T), prolactin (PRL) and progesterone (Р4) were assessed in 41 tumor samples obtained during operative therapy of patients with endometrial cancer T1-2N0M0 in 10% homogenates with enzyme-linked immunosorbent assay (ELISA) with the use of standard test systems (XEMA). Mean age was 53.4±3.2 years, histological structure - endometrioid adenocarcinoma. IgG antibodies against herpes simplex virus (HSV), cytomegalovirus (CMV) and chlamydias were detected in tumor tissue with ELISA. The polymerase chain reaction method was used for the detection of CMV, HSV and chlamydia infection in ovarian tissue. Adenocarcinoma without IgG against infectious agents served as a control. Results: IgG against HSV antigens were found in 26.8% of the cases; IgG against CMV+HSV antigens+clamydias – in 17.1%; IgG against CMV antigens and IgG against CMV+HSV antigens – in 12.2%; 31.7% of the tissues did not have IgG against the infections. It was found that the infections alone or in combinations resulted in the increase of Е1 level by 1.3-1.7 times, Е2 by 1.6-1.9 times, PRL by 3 times while T concentration decreased by 1.4-3.4 times in comparison with adenocarcinomas without infectious agents. Р4 concentration in infected tumors did not differ significantly from the indices in uninfected ones remaining reduced in comparison with intact endometrium tissue. Conclusions: Infections in tumor tissue, alone or in combinations, cause additional potential for changes and precarious states that can result in drug resistance, resistance to treatment, and reduction of differentiation, that is an increase of malignancy. Disclosure: All authors have declared no conflicts of interest.

Clinical application of viral cerebrospinal fluid PCR testing for diagnosis of central nervous system disorders: a retrospective 11-year experience

The cerebrospinal fluid (CSF) polymerase chain reaction (PCR) is the gold standard to detect cerebral viral activity. As positive findings do not prove an impact on the neurological disorder, data interpretation is difficult. To better assess the impact of positive CSF PCR findings in different neurological diseases and to identify coherences facilitating CSF PCR data interpretation, we performed this retrospective analysis of CSF PCR data of 481 pediatric and 2604 adult patients, including herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and enteroviruses (EV). Nucleic acid of EBV was detected in 1.6% (39/2449), of VZV in 1.3% (34/2624), of HSV in 1.24% (37/2994), of EV in 0.4% (10/2364), of HHV-6 in 0.17% (4/2417), and of CMV in 0.2% (5/2514) of the patients. Newborns and elderly people showed highest infection rates. HSV, VZV, and EV prevailed in typical infectious central nervous system (CNS) diseases; EBV, in further inflammatory neurological diseases; HSV and EBV, in immunocompromised patients; and EBV, HSV, and HHV-6, in further non-inflammatory neurological diseases. Analysis of successive PCR studies revealed delayed viral detection for EBV (6/147) and HSV (1/217), respectively. Rapid viral clearance was typical for HSV, VZV, CMV, and EV infections, although the maximum duration of viral detection was 15days for HSV and 12days for VZV, respectively. This suggests that the detection of HSV, VZV, CMV, and EV strongly indicates symptomatic viral CNS disease. Secondary viral reactivation mostly underlies positive EBV and HHV-6 findings. Their detection does not rule out clinical impact but recommends searching for additional underlying conditions.

Clinical validation of the Lyra direct HSV 1+2/VZV assay for simultaneous detection and differentiation of three herpesviruses in cutaneous and mucocutaneous lesions.

We evaluated the Lyra Direct HSV 1+2/VZV multiplex real-time PCR assay for the detection and differentiation of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) on 695 consecutive cutaneous and mucocutaneous lesion specimens. The intra-assay and interassay coefficient of variation values for the Lyra assay were 0.29 to 1.30% and 2.33 to 2.61%, respectively. The sensitivities, specificities, and positive and negative predictive values were 93.4 to 95.0%, 96.1 to 96.8%, 78.0 to 80.3%, and 99.0 to 99.1%, respectively, in comparison to those of viral culture. The values were further improved when a resolution analysis was performed with a laboratory-developed PCR assay.

HSV oropharyngeal shedding among HIV-infected children in Tanzania.

Herpes simplex virus (HSV) oral shedding has not been studied among HIV-positive children in Africa. We sought to evaluate longitudinal oral HSV reactivation in HIV-positive and -negative children. Twenty HIV-positive antiretroviral-naive and 10 HIV-negative children aged 3-12 years in Tanzania were followed prospectively for 14 days. Oral swabs were collected daily and submitted for HSV DNA PCR analysis. Clinical data were collected via chart review and daily diaries. HSV DNA was detected in 10 (50%) of HIV-positive and 4 (40%) of HIV-negative children. Children who shed HSV had virus detected in a median of 21.4% of samples; shedding was intermittent. Median CD4 count among HIV-infected children was 667 cells/µL in those with positive HSV DNA and 886 cells/µL in those who were negative (p = 0.6). Of the HIV-positive children reporting prior sores, five (83%) had positive HSV swabs, whereas the one HIV-negative child with prior sores did not have a PCR-positive swab. HSV is detected frequently in children with and without HIV. HIV-infected children reporting oral sores have a high rate of HSV detection. Given the proven strong interactions between HIV and HSV, further study of co-infection with these viruses is warranted in children.

Diagnosis of genital herpes simplex virus infection in the clinical laboratory.

Since the type of herpes simplex virus (HSV) infection affects prognosis and subsequent counseling, type-specific testing to distinguish HSV-1 from HSV-2 is always recommended. Although PCR has been the diagnostic standard method for HSV infections of the central nervous system, until now viral culture has been the test of choice for HSV genital infection. However, HSV PCR, with its consistently and substantially higher rate of HSV detection, could replace viral culture as the gold standard for the diagnosis of genital herpes in people with active mucocutaneous lesions, regardless of anatomic location or viral type. Alternatively, antigen detection—an immunofluorescence test or enzyme immunoassay from samples from symptomatic patients--could be employed, but HSV type determination is of importance. Type-specific serology based on glycoprotein G should be used for detecting asymptomatic individuals but widespread screening for HSV antibodies is not recommended. In conclusion, rapid and accurate laboratory diagnosis of HSV is now become a necessity, given the difficulty in making the clinical diagnosis of HSV, the growing worldwide prevalence of genital herpes and the availability of effective antiviral therapy.

Detection and Differentiation of Herpes Simplex Viruses by Use of the Viper Platform: Advantages, Limitations, and Concerns

The Viper HSV-Q(x) assay was evaluated for the detection of herpes simplex virus 1 (HSV-1) and HSV-2 in specimens from oral, anogenital, and other miscellaneous sites. The HSV-Q(x) assay was found to be highly sensitive and accurate; however, a gray zone may be required for specimens with values falling between 50 and 800 maximum relative fluorescence units.

Virologic and Immunologic Evidence of Multifocal Genital Herpes Simplex Virus 2 Infection

Genital herpes simplex virus (HSV) reactivation is thought to be anatomically and temporally localized, coincident with limited ganglionic infection. Short, subclinical shedding episodes are the most common form of HSV-2 reactivation, with host clearance mechanisms leading to rapid containment. The anatomic distribution of shedding episodes has not been characterized. To precisely define patterns of anatomic reactivation, we divided the genital tract into a 22-region grid and obtained daily swabs for 20 days from each region in 28 immunocompetent, HSV-2-seropositive persons. HSV was detected via PCR, and sites of asymptomatic HSV shedding were subjected to a biopsy procedure within 24 h. CD4(+) and CD8(+) T cells were quantified by immunofluorescence, and HSV-specific CD4(+) T cells were identified by intracellular cytokine cytometry. HSV was detected in 868 (7%) of 11,603 genital swabs at a median of 12 sites per person (range, 0 to 22). Bilateral HSV detection occurred on 83 (67%) days with shedding, and the median quantity of virus detected/day was associated with the number of sites positive (P < 0.001). In biopsy specimens of asymptomatic shedding sites, we found increased numbers of CD8(+) T cells compared to control tissue (27 versus 13 cells/mm(2), P = 0.03) and identified HSV-specific CD4(+) T cells. HSV reactivations emanate from widely separated anatomic regions of the genital tract and are associated with a localized cellular infiltrate that was demonstrated to be HSV specific in 3 cases. These data provide evidence that asymptomatic HSV-2 shedding contributes to chronic inflammation throughout the genital tract. This detailed report of the anatomic patterns of genital HSV-2 shedding demonstrates that HSV-2 reactivation can be detected at multiple bilateral sites in the genital tract, suggesting that HSV establishes latency throughout the sacral ganglia. In addition, genital biopsy specimens from sites of asymptomatic HSV shedding have increased numbers of CD8(+) T cells compared to control tissue, and HSV-specific CD4(+) T cells are found at sites of asymptomatic shedding. These findings suggest that widespread asymptomatic genital HSV-2 shedding is associated with a targeted host immune response and contributes to chronic inflammation throughout the genital tract.

Light Microscopy, Culture, Molecular, and Serologic Methods for Detection of Herpes Simplex Virus

Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) cause a variety of human diseases, ranging from acute to chronic and mild to severe. The absence of curative therapy results in lifelong carriage marked by recurrent outbreaks and allows transmission of the virus to uninfected individuals. Nonspecific lesions, variable presentation, and chronic carriage necessitate the use of different laboratory testing methods appropriate for each presentation. A thorough understanding of the performance characteristics and limitations of available tests is critical for selection of the appropriate test and interpretation of results. Clinical sensitivity, specificity, and selection of the appropriate methodology is paramount to avoid misdiagnosis and guide therapy. In this article we review the different methods for detection and typing of HSV, including light microscopy, culture, serology, and nucleic acid-based tests. We discuss the strengths and weaknesses of each method for diagnosing HSV infection, cite performance characteristics, and review appropriate clinical uses.

Comparison of Simplexa HSV 1 &amp; 2 PCR with Culture, Immunofluorescence, and Laboratory-Developed TaqMan PCR for Detection of Herpes Simplex Virus in Swab Specimens

The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type.

P5.080 Comparison of an In-House Polymerase Chain Reaction and Direct Fluorescent Antibody Assay For Detection and Typing of Herpes Simplex Virus in Clinically Suspected Genital Herpes

BackgroundMolecular assays based on PCR have become an important tool for the detection of herpes simplex virus-1&2 DNA in clinical specimens. Detection and typing of HSV can also be done...

P2.036 Detection of Herpes Simplex Viruses 1 and 2 from Clinical Samples with a Fully-Automated PCR Test on the Cobas ® 4800 System

BackgroundIdentification of genital herpes can have important implications for clinical management of HIV infected patients, immunosuppressed individuals, pregnant women, and individuals with HSV seronegative partners. This study was performed to...

Etiology of Genital Ulcer Disease in a Sexually Transmitted Infection Reference Center in Manaus, Brazilian Amazon

ObjectivesTo determine the etiology and factors associated with genital ulcer disease (GUD) among patients presenting to a sexually transmitted infections clinic in Manaus, Brazil; and to compare a multiplex polymerase chain reaction (M-PCR) assay for the diagnosis of GUD with standard methods.MethodsUlcer swabs were collected and used for Tzanck test and processed in an M-PCR to detect herpes simplex virus (HSV-1/2), Treponema pallidum (T. pallidum), and Haemophilus ducreyi (H. ducreyi). Sera were tested for HIV and syphilis antibodies. Multivariable analysis was used to measure the association between clinical aspects and GUD. M-PCR results were compared with syphilis serology and Tzanck tests.ResultsOverall, 434 GUD samples were evaluated, 84.8% from men. DNA from HSV-2 was detected in 55.3% of GUD samples, T. pallidum in 8.3%, HSV-1 in 3.2%, and 32.5% of GUD specimens were negative for the DNA of all three pathogens. No cases of H. ducreyi were identified. HIV serology among GUD patients was 3.2%. Treponemal antibodies and Tzanck test positivity for genital herpes was detected in 25 (5.8%) and in 125 (30.3%) of GUD patients, respectively. In multivariable analysis genital herpes etiology by M-PCR was associated with the vesicular, multiple and recurrent lesions whereas T. pallidum with non-vesicular, non-recurrent lesions. Compared to M-PCR, syphilis serology was 27.8% sensitive and 96.2% specific whereas Tzanck test was 43.8% sensitive and 88.9% specific.ConclusionsThe predominance of genital herpes etiology suggests a revision of existing national syndromic treatment guidelines in Brazil to include antiherpetic treatment for all GUD patients. The use of M-PCR can significantly improve the diagnosis of GUD and provide a greater sensitivity than standard diagnostics.

The development of a multiplex real-time PCR for the detection of herpes simplex virus 1 and 2, varizella zoster virus, adenovirus and Chlamydia trachomatis from eye swabs

Infectious conjunctivitis can be difficult to distinguish clinically due to the considerable overlap in clinical presentation so clinical diagnosis of conjunctivitis is often insufficient. It is therefore necessary to have a rapid diagnostic test that differentiates between the different causes of infectious conjunctivitis. Screening clinical samples by sample type/syndrome based multiplex real time PCR would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient.A multiplex real-time PCR assay for rapid and simultaneous detection of HSV 1 and 2, VZV, adenovirus and Chlamydia trachomatis (C. trachomatis) from eye swabs was developed and evaluated. The multiplex assay was shown to be sensitive, specific and robust. Reductions in sample turn around times have been achieved by reducing the amount of separate tests needed to be carried out.

Delays in Initiation of Acyclovir Therapy in Herpes Simplex Encephalitis

Diagnosis of herpes simplex encephalitis (HSE) is based on clinical findings, MRI, and detection of herpes simplex virus (HSV) DNA in cerebrospinal fluid (CSF) using polymerase chain reaction amplification. Delays in starting treatment are associated with poorer clinical outcomes. We assessed the timing of initiation of acyclovir therapy in HSE. Inpatient databases from seven hospitals in Winnipeg, Manitoba were used to identify individuals diagnosed with encephalitis and HSE from 2004 to 2009. The time taken to initiate therapy with acyclovir and the reasons for delays were determined. Seventy-seven patients were identified; 69 (90%) received acyclovir; in the others a non-HSV infection was strongly suspected. Thirteen patients were subsequently confirmed to have HSE. Acyclovir was initiated a median of 21 hours (3-407) after presentation in encephalitis cases, and a median of 11 hours (3-118) in HSE. The most common reason for delay was a failure to consider HSE in the differential diagnosis, despite suggestive clinical features. Where therapy was delayed in HSE patients, the decision to begin acyclovir was prompted by transfer of the patient to a different service (55%), recommendations by consultants (18%), imaging results (18%), and CSF pleocytosis (9%). Delays in initiating acyclovir for HSE are common, and are most often due to a failure to consider HSE in a timely fashion on presentation. In order to improve patient outcomes, physicians should be more vigilant for HSE, and begin acyclovir therapy expeditiously on the basis of clinical suspicion rather than waiting for confirmatory tests.

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence Detector

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH).

Near instrument-free, simple molecular device for rapid detection of herpes simplex viruses

The first near instrument-free, inexpensive and simple molecular diagnostic device (IsoAmp® HSV, BioHelix Corp., MA, USA) recently received US FDA clearance for use in the detection of herpes simplex viruses (HSV) in genital and oral lesion specimens. The IsoAmp HSV assay uses isothermal helicase-dependent amplification in combination with a disposable, hermetically-sealed, vertical-flow strip identification. The IsoAmp HSV assay has a total test-to-result time of less than 1.5 h by omitting the time-consuming nucleic acid extraction. The diagnostic sensitivity and specificity are comparable to PCR and are superior to culture-based methods. The near instrument-free, rapid and simple characteristics of the IsoAmp HSV assay make it potentially suitable for point-of-care testing.

Comparative clinical evaluation of the IsoAmp® HSV Assay with ELVIS® HSV culture/ID/typing test system for the detection of herpes simplex virus in genital and oral lesions

BackgroundThe novel IsoAmp® HSV Assay employs isothermal helicase-dependent nucleic acid amplification and a user-friendly disposable test device to achieve rapid (<1.5h), on-demand qualitative detection of herpes simplex virus (HSV) types 1 and 2 in oral and genital lesions. ObjectivesTo compare performance of the IsoAmp® HSV Assay with the ELVIS® HSV ID/typing (shell-vial culture and DFA) test system for clinical specimens collected from oral and genital lesions in symptomatic patients. Study designA total of 994 specimens from male and female genital and oral lesions were obtained and evaluated at five study sites in the United States. Results from the IsoAmp® HSV Assay were compared to those from the ELVIS® system. Separate reproducibility studies were performed at 3 sites using a blinded and randomized study panel. Discrepant specimens were resolved by bidirectional sequencing analysis. ResultsAfter discrepant analysis, overall agreement of IsoAmp® with ELVIS® was 98.8% with 37.0% overall prevalence (all study sites). Reproducibility rates were well within expectations. ConclusionThe IsoAmp® HSV Assay showed excellent performance for clinical use for detection of HSV in genital and oral specimens. In contrast to ELVIS®, IsoAmp® HSV offers excellent sensitivity plus rapid on-demand testing and simpler specimen preparation.

Herpes simplex virus encephalitis despite normal cell count in the cerebrospinal fluid*

To describe herpes simplex virus encephalitis despite normal cell count in the cerebrospinal fluid in patients with malignoma after whole brain irradiation. Blood and cerebrospinal fluid analysis and magnetic resonance imaging. Three male and two female patients with malignoma and a recent history of whole-brain irradiation presented with impaired consciousness with or without epileptic seizure. Although cerebrospinal fluid analysis revealed a normal cell count, herpes simplex virus DNA was detected in all samples by polymerase chain reaction. In patients with impaired consciousness, epileptic seizure, or temporal lobe symptoms of new onset and a recent history of brain irradiation with normal cerebrospinal fluid, an atypical anergic course of herpes simplex virus encephalitis should be considered. Herpes simplex virus polymerase chain reaction should be used as method of choice to detect herpes simplex virus genomes as early as possible rather than relying on routine cerebrospinal fluid parameters. Importantly, antiviral therapy should be started without delay in any case of faint suspicion and should be continued until herpes simplex virus encephalitis is clearly ruled out.