Progressive herpes simplex virus culture

Therefore, if typing is not done routinely, the isolate should be saved until it is determined whether typing is required or not. The reporting of type-specific HSV will aid the clinician in counselling and management of the patient. Viral antigen detection may be a suitable alternative to culture for smaller laboratories in which the expense of maintaining cell lines is unwarranted.

Antigen detection is also an alternative where specimen handling and transportation conditions could inactivate any virus present. This could occur, for example, in laboratories serving remote locations with prolonged specimen transportation times under uncertain conditions. For detecting HSV in lesions, the sensitivity of antigen detection tests may be the same as or greater than that of culture 24 , Detection of HSV antigens has been achieved in fixed cells by DFA tests or immunoperoxidase tests on fixed, solubilized cell specimens 24 - These methods can give a useful result even in the absence of cultivable virus.

The demonstration of the presence of HSV antigen by DFA staining of smears can provide a rapid adjunct to cell culture. Although the slide may be prepared by the clinician, it is ideally prepared by the laboratory using a cytospin method and a swab specimen collected as described earlier.

Staining of the slide is as directed by the manufacturer of the fluorescein-labelled antibody. The slide is examined using a fluorescence microscope, with a positive test indicated by the presence of a characteristic pattern of apple-green fluorescence in the nucleus and cytoplasm of the basal and parabasal cells.

Only intact cells should be examined. An inconclusive result may be obtained if fewer than 50 intact cells are present on each well. HSV infection causes typical cytopathic changes in genital epithelial cells 3. The cells become enlarged, with intranuclear inclusions, often with the formation of multinucleated cells. Prepared slides are stained with a Wright-Giemsa stain and then examined under light microscopy. Hematoxylin and eosin or the Papanicolaou stains may also be used.

This test can be performed when an urgent result is needed and no alternative test is immediately available, but it does not negate the need for follow-up testing of all negatives with a more sensitive test. Direct examination of vesicle fluid or other clinical material by electron microscopy for the diagnosis of HSV is limited by the fact that viral morphology cannot be used to distinguish HSV from other herpes viruses eg, varicella zoster virus Viral DNA may be detected by hybridization techniques using radiolabelled or biotinylated probes 27 , These methods have largely been superceded by more sensitive and less laborious procedures which utilize amplification of the target HSV DNA by polymerase chain reaction PCR.

Specificity of the amplification method is assured by either undertaking a second PCR with target-specific primers nested PCR or by HSV-specific probe hybridization of amplified products. The majority of laboratories have confined their use of methods such as PCR to the investigation of suspected HSV encephalitis In this situation, the enhanced sensitivity over culture- or antigen-based procedures is well-recognized, and the clinical value of positive results is clearly demonstrable.

In the case of possible genital herpes, PCR detects viral DNA for several days after lesions do not contain demonstrable infectious virus This may mean that a laboratory switching to sensitive procedures based on nucleic acid amplification may have an increased number of positive results on lesion samples with possible clinical dilemmas regarding the relevance of positive results obtained after treatment. Although PCR can detect HSV DNA from later stages of lesions than virus culture, there is a theoretical risk of false-positive results occurring due to sample contamination before amplification.

Laboratories undertaking PCR-based procedures need to have separate areas and equipment for pre- and postamplification handling of specimens to minimize this kind of problem.

Samples giving discordant results eg, positive by PCR and negative on culture are usually confirmed by a second PCR directed to a different gene to ensure assay specificity. As with other molecular diagnostic tests, the sensitivity of PCR is much greater than the gold standard of culture 31 - The advent of real-time PCR systems, where products are detected in a closed-tube system without any post-amplification handling, has minimized the risk of false-positive results by PCR.

While the equipment to undertake real-time PCR is still relatively expensive, the small reaction volumes and minimal technical hands-on time particularly when kit-based reagents are used make these methods very cost effective for many laboratories. The detection of antibodies to HSV allows for diagnosis when other virological methods cannot be performed or yield negative results It is particularly useful in identifying the asymptomatic carrier of infection because, as discussed above, the majority of transmission occurs while the person is asymptomatic.

Thus far, the use of these tests has largely been confined to seroepidemiological studies and case management for HSV, while specific clinical uses for serological testing remains a much debated topic.

Clinical, virological and serological classification of infection with genital herpes simplex virus HSV. Serological assays that are not type-specific have limited clinical utility. In addition, no serological test is able to differentiate between oral and genital infection with HSV. This difference has been exploited in developing type-specific serological tests.

A recent review describes the new HSV type-specific antibody tests Finally, it appears that seroreversion or waning of immune response to gG-2 occurs with time, raising concerns about the long-term reliability of these tests This test is expensive, time consuming and requires skilled interpretation. When initial results are indeterminate or atypical, adsorption of sera with type-specific antigen and reblotting can sometimes 'clean up' the blot and improve interpretation.

Although most of the available literature evaluating the performance of type-specific tests was based on kits developed by Gull Laboratories USA , these tests have now been withdrawn from the market. Presently, two companies produce four kits for the diagnosis of HSV type-specific antibodies. A number of antiviral agents have been developed for the management of HSV infections; of these, acyclovir is the most commonly used. Resistance of HSV to acyclovir has become increasingly common, with almost all clinically significant acyclovir-resistant strains seen in immunocompromised patients, especially those coinfected with HIV 43 , The development of resistance usually results from mutations within the viral genome, and the presence of selective drug pressure usually results in the emergence of a resistant virus population.

The isolation of HSV from persisting lesions despite adequate dosages and blood levels of acyclovir should raise the suspicion of acyclovir resistance. The antiviral activity of acyclovir requires an initial phosphorylation step by the viral enzyme thymidine kinase TK 45 , Two subsequent phosphorylation steps are mediated by cellular kinases. The resulting triphosphorylated acyclovir then specifically inhibits herpesvirus DNA polymerases.

Three different mechanisms of resistance of HSV to acyclovir have been identified. The most common is found in viruses that lack a functional TK TK - mutants and, thus, are unable to monophosphorylate acyclovir.

Less commonly, some resistant viruses produce a functional TK enzyme that is unable to phosphorylate acyclovir because of altered substrate specificity TK A mutants. Foscarnet directly inhibits herpesvirus DNA polymerases and resistance develops because of altered viral DNA polymerases. Vidarabine resistance also occurs rarely. Vidarabine is phosphorylated by cellular enzymes and then inhibits virally encoded DNA polymerase. The complexity of drug sensitivity assays for antiviral resistance limits their availability.

At the present time, they are only performed by specialized laboratories. Susceptibility testing of strains of HSV against various antiviral agents is usually performed in the laboratory using modifications of one of the following: plaque reduction assays, dye uptake assays or DNA hybridization assays 45 , The plaque reduction assay was the first antiviral susceptibility testing method performed to determine the susceptibility of viruses to antiviral agents and is the standard against which other tests are compared.

These tests are time consuming and may soon be replaced by genotypic tests that can be processed more quickly. All laboratories providing diagnostic services for the detection of HSV in clinical samples or performing HSV serological assays must participate in the testing of proficiency panels provided by external agencies whenever possible for all tests performed. If proficiency testing for specific assays is not available eg, HSV DNA detection in swab material or type-specific serological testing , then specimen exchange among laboratories performing such testing should be arranged as an alternative form of proficiency testing.

Subpassages of HSV clinical isolates should be inoculated with each batch of HSV roller tube or shell vial cultures to serve as positive controls. Uninfected tubes or shell vial cultures serve as negative controls. Both infected and uninfected cell monolayers should be observed for the presence or absence of HSV CPE and stained to observe for typical immunofluorescence with HSV monoclonal antibodies. Positive controls should exhibit characteristic CPE and immunofluorescence with type-specific antisera, while negative controls should not.

Variations in sensitivity may occur in cultured cell lines for various reasons. Positive and negative control slides should be included daily in each run to ensure that the antibody reagents are performing correctly. Typical immunofluorescence should be observed in the positive controls but not the negative controls. Positive and negative controls must be included with each batch of sera tested.

Order This Test. Useful For Suggests clinical disorders or settings where the test may be helpful. Method Name A short description of the method used to perform the test. Reporting Name Lists a shorter or abbreviated version of the Published Name for a test. Aliases Lists additional common names for a test, as an aid in searching. Specimen Type Describes the specimen type validated for testing. Specimen Required Defines the optimal specimen required to perform the test and the preferred volume to complete testing.

Specimen Minimum Volume Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory. Stool: 5 g Urine: 0. Reject Due To Identifies specimen types and conditions that may cause the specimen to be rejected. Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included.

Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test. Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this. No virus isolated. Interpretation Provides information to assist in interpretation of the test results.

Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances. Clinical Reference Recommendations for in-depth reading of a clinical nature. Curr Infect Dis Rep Nov ;11 6 2. J Clin Virol ;17 1 3. J Infect Dis Feb 1; 3 5. J Clin Virol May;30 1 6. Method Description Describes how the test is performed and provides a method-specific reference. More blood tests will likely be needed to confirm the diagnosis or the exact cause.

If the culture is positive for herpes, you may have recently become infected. You may have become infected in the past and are currently having an outbreak. Specimen collection and handling for diagnosis of infectious diseases. St Louis, MO: Elsevier; chap Dermatologic therapy and procedures. Lookingbill and Marks' Principles of Dermatology. Philadelphia, PA: Elsevier; chap 4. Herpes simplex virus infections. Goldman-Cecil Medicine.

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