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Assay Sheet

Test ID

8500 Herpes Simplex Virus Type 1 & 2 Real-time qPCR

CPT Code

87530 

Clinical Utility

HSV-1 and HSV-2 are well known etiologic agents of gingivostomatitis, conjunctivitis, keratitis, herpetic whitlow,  and genital infections as well as encephalitis, aseptic meningitis, atypical pneumonias and a wide variety of other clinical conditions. Quantitative HSV DNA PCR is the ideal diagnostic tool for CNS specimens, since HSV will rarely grow from CSF in cell culture. Quantitative PCR can be used to detect the presence of the virus as well as track the course of infections and response to treatment.

Procedure

Extraction of Herpes Simplex viral DNA from plasma, CSF, bone marrow, or other biological fluids or tissues followed by amplification and detection using real-time, quantitative PCR. An internal control is added to ensure the extraction was performed correctly and the PCR reaction was not inhibited.

Specimens

Whole Blood: 3 to 5 ml collected in EDTA (lavender top) tube. Do not freeze; ship ambient. Testing will be performed on plasma separated from the submitted whole blood specimen. Whole blood specimens are accepted as a matter of convenience for the originating laboratory. 

Plasma: 3 to 5 ml separated from whole blood collected in EDTA (lavender top) tube; ship ambient.

Amniotic Fluid: 1 ml minimum, submitted in sterile, screw-top tube; ship ambient.

Bone Marrow: 2 ml minimum, collected in an EDTA (lavender top) tube. Do not freeze.

Bronchial Lavage/Bronchial Wash: 1 to 3 ml, collected in sterile, screw-cap tube; ship ambient.

Conjunctival/Eye Swab: Swab the conjunctiva with sterile swab and place in 1 to 2 ml sterile saline, M4, or viral transport media; ship ambient.

CSF: 1 ml minimum, submitted in sterile, screw-cap tube; ship on dry ice.

Genital Swab: Sterile swab placed in 1 to 2 ml of sterile saline, M4, or viral transport media submitted in a sterile, screw-cap tube; ship ambient.

Pleural Fluid: 1 ml minimum, submitted in a sterile, screw-top tube; ship ambient.

Skin Swab: Sterile swab placed in 1 to 2 ml sterile saline, M4, or viral transport media in a sterile, screw-cap tube; ship ambient. Do not use calcium alginate swab or wood shafted swab.

Throat Gargle:  Instruct patient to gargle with 2 to 3ml sterile saline.  Expectorate into sterile cup, then transfer contents to sterile, screw-cap tube for shipment; ship ambient.  

Tissue: Place in a sterile, screw-cap tube, add a small amount of saline to keep moist. Prefer 1 mm x 1 mm specimen. Prefer fresh over formalin fixed for maximum sensitivity; ship ambient.

Upper respiratory aspirate (NP aspirate, nasal aspirate, tracheal aspirate, etc.): Instill 1 to 2 ml sterile saline into desired location and gently aspirate contents. Place collected fluid into sterile screw-cap tube; ship ambient.

Upper respiratory swab (NP swab, throat swab): Swab desired location with sterile, flexible shaft swab, preferably a flocked swab. Place swab into 1 to 2 ml sterile saline, M4, or viral transport media in sterile screw-cap tube. Do not use calcium alginate swab or wood shafted swab; ship ambient.

Urine: 1 to 2 ml sample collected in a sterile urinalysis container. Transfer to a 15 ml sterile screw-cap tube; ship ambient.

Vitreous Fluid: Place collected vitreous fluid into small, sterile, screw-cap tube; ship ambient.

Other specimens may be accepted for testing; however the following comment will appear in the final report: "The clinical utility of this result has not yet been demonstrated in the peer reviewed literature and is therefore unknown." Call ViraCor for further information.

Causes for rejection

Whole blood frozen, wood shafted swab, calcium alginate swab. Call ViraCor at 800-305-5198 if specimen is greater than 96 hrs old.

Specificity

The HSV-1 and HSV-2 primers and probes used in this assay are specific for HSV-1 & 2 only and detect known HSV-1 & 2 strains based on similarity search algorithms. The individual primers and probes for HSV-1 and HSV-2 are unique and specific, with no cross reactivity between the HSV-1 and HSV-2 primers. Additionally, no cross reactivity was detected when tested against adenoviruses, BKV, CMV, EBV, HHV-6 variant A, HHV-6 variant B, HHV-7, HHV-8, JCV, parvovirus B19, SV-40, and VZV.

Herpes Simplex Virus Type 1 & 2 Assay Range

100 copies/ml to 1 x 10¹⁰ copies/ml

The assay differentiates between HSV-1 and HSV-2.

Turnaround Time

Same day (within 8 to 12 hours of receiving specimen), Monday through Saturday

Shipping

Pathogen Overview

ABOUT HERPES SIMPLEX VIRUS 1 & 2

The two herpes simplex viruses, HSV-1 and HSV-2, are linear, double-stranded DNA viruses with an icosahedral capsid. Both are members of the Herpesviridae family and Alphaherpesvirinae subfamily, along with VZV. The herpesviruses and resulting infections were first identified and demonstrated in the early 1900s. All three viruses share the ability to establish latency in their host following primary infection. HSV-1 and HSV-2 establish latency in neurons of dorsal root ganglia.

Herpes simplex virus (HSV) infections are extremely common in humans; in most cases, they are simply bothersome and frustrating. However, infections can become life-threatening for some individuals, especially those who are immunocompromised. Lethal, disseminated disease can occur in neonates if the virus is acquired during passage through an infected birth canal or through prolonged rupture of the membranes in an infected mother.

HERPES SIMPLEX VIRUS 1 & 2 CLINICAL MANIFESTATIONS

Humans are the only natural carriers of HSV, which is transmitted via of direct contact with secretions that are infected with the virus. Viral titers are higher when the infection is actively producing lesions; it is believed that risk of transmission is greater during this time, although the virus has been documented to be transmitted during periods of apparent latency. At any given time, it is estimated that HSV can be isolated from 1 to 5% of asymptomatic adults and 20% of asymptomatic children. Primary infections with HSV-1 most commonly involve the mouth and throat. Primary infections with HSV-2 most commonly involve the genital region; however, both viruses can infect any mucous membrane. As orogenital sexual practices have become more common, it is not unusual to see genital infections caused by HSV-1. Autoinoculation between different areas of the body, such as the eye, also occur.

Primary HSV-1 infections are often asymptomatic, but can manifest as gigivostomatitis or pharyngitis, most commonly in children under age 5. Probability and age of acquisition are inversely related to socioeconomic status. In more affluent societies, a second wave of acquisition by exchange of salivary secretions is common during adolescence. A 2 to 12 day incubation period is followed by fever, sore throat, and pharyngeal edema with erythema. Vesicles develop on the mucosa of the mouth and throat, which ulcerate and proliferate, often spreading to the soft palate, tongue, and floor of the mouth. Lesions may appear on the face and gums and are usually tender and bleed easily. Fever, toxicity, and severe pain may linger for several days, but the infection usually runs its course within 10 to 14 days. Encephalitis is an unusual manifestation of HSV; HSV-1 is most often responsible, except in the case of neonates. If left untreated, the case fatality rate is approximately 70%, with survivors suffering significant neurologic sequelae.

Primary HSV-2 infections are also often asymptomatic, but significant disease often occurs, particularly in females. Genital HSV infections are manifested as ulcerating vesicular lesions on the vulva, vagina, cervix, urethra, and perineum in females, and on the penis in males and the rectum and perianal region in homosexual males. Symptoms can include inguinal lymphadenopathy, pain, itching, swelling, discharge, and dysuria; systemic symptoms include fever and malaise. Meningitis also occurs in about 10% of cases. Initial disease will often be less severe if antibodies are present from HSV-1.

Following initial infection, a lifetime viral latency is established in nearby nerve ganglia. Reactivation occurs with varying frequency, depending upon the individual. Reactivation is often preceded by a prodrome consisting of hyperesthesia.

Immunocompromised patients have a greater risk of developing severe, disseminated HSV infections. Immunocompromised patients can include solid organ transplant patients, bone marrow transplant patients, HIV patients, burn victims, and neonates. Transplant patients often excrete HSV in throat washings during the initial weeks following transplantation. For this reason, transplant patients are often prophylaxed with acyclovir. Typical infections are respiratory or gastrointestinal in nature and involve tracheobronchitis, pneumonia, esophagitis, and hepatitis.

Neonatal herpes is a very serious disease; prior to the use of acyclovir, it was often fatal. Infants acquire neonatal herpes by passing through an infected birth canal or through prolonged rupture of the membranes in an infected mother. If the mother has a primary infection at the time of delivery, the neonate has a 30 to 40% risk of acquiring the virus. If the mother has a recurrent infection at the time of birth, the risk of neonate infection is much lower, around 3 to 4%. The viral infection can be manifested as encephalitis or disease localized to mucocutaneous surfaces, such as skin, eyes, and mouth. If untreated, neurological disease carries a high fatality rate, with survivors experiencing severe neurological sequelae. Local disease in the neonate can progress to severe disseminated disease, if left untreated.

HERPES SIMPLEX VIRUS 1 & 2 LABORATORY DIAGNOSIS

Culture of HSV was the mainstay of diagnosis for many years, as the virus grows easily in a wide variety of cell lines in vitro. However, the temperature lability of HSV was a significant limitation of culture. If the time delay between collection and arrival in the laboratory was too great, the virus would no longer be infectious, resulting in a false negative culture. In addition, HSV will not grow from CSF samples, yet another significant limitation of culture. With the advent of molecular detection methods, analysis of CSF for HSV DNA quickly became the preferred method of diagnosis when ruling out HSV meningitis. Real-time, quantitative polymerase chain reaction (PCR) has become a powerful diagnostic tool for many clinical situations, since it allows determination of a patient’s viral burden (viral load) in a given sample. As a result, clinicians are able to track response to interventions, such as administration of acyclovir.

HERPES SIMPLEX VIRUS 1 & 2 TREATMENT

Acyclovir has been used in the treatment of HSV infections for about 30 years. It is highly specific for herpes virus-infected cells and has an acceptable safety profile. The development of valacyclovir offers similar clinical and safety benefits, with a simpler treatment regimen. Acyclovir and valacyclovir have been studied for the treatment of HSV infections in bone marrow and renal transplant patients, as well as individuals with HIV. Both drugs have been effective in each of these groups. In immunocompromised patients, acyclovir is helpful for both treatment and suppression of recurrent lesions. This is true for transplant recipients, leukemics undergoing chemotherapy, and AIDS patients. Acyclovir has also proven invaluable in treatment of neonates with HSV infections.

Selected References

Griffiths PD. Tomorrow’s challenges for herpesvirus management: potential applications of Valacyclovir. J Infect Dis. 2002;186
(suppl 1):S131-S137.

Hirsch MA. Herpes simplex virus. In: Mandell GL, Bennett JE, Dolin, eds. Mandell, Douglas and Bennett’s Principles and Practice of
Infectious Diseases. Vol 2. 4th ed. New York, NY: Churchill Livingstone; 1995:1336-1345.

Knipe D, Howley P. Fields Virology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2006.

Simmons A. Clinical manifestations and treatment considerations of herpes simplex virus infection. J Infect Dis. 2002;186(suppl 1):S71-S77.

Tang Y-W, Mitchell PS, Espy MJ, Smith TF, Persing DH, et al. Molecular diagnosis of herpes simplex virus infections in the central nervous system. J Clin Microbiol. 1999;(37):2127-2136.

Whitley RJ, Roizman B. Herpes simplex virus infections. Lancet. 2001;(357):1513-1518.

PAO-10-0707 PCR tests are performed pursuant to a license agreement with Roche Molecular Systems, Inc.

Abstracts & Publications

Corey L. Challenges in genital herpes simplex virus management. J Infect Dis. 2002;186(suppl 1)S29-S33.

Development of serologic assays to detect antibodies to herpes simplex virus (HSV) glycoproteins (g)G1 and (g) G2 has allowed accurate definition of the seroprevalence of HSV-2 worldwide. Studies from all continents indicate epidemic proportions of HSV-2 infection. In the United States, 1 in 5 sexually active adults is infected. In Africa and the Caribbean, HSV prevalence is higher. Since the development of the acyclic nucleoside derivatives acyclovir, famciclovir, and valacyclovir, treatment of mucocutaneous HSV is a practice of everyday medical care. Yet, despite effective drugs, there is widespread discontent by clients and providers about care of patients with genital herpes. Much of this relates to transmission complexities and the varied natural history of the infection. However, over time, most patients adjust to their disease and the medical and psychosocial complications. Recent studies show condoms reduce transmission, providing an important tool for counseling the patient with newly diagnosed genital herpes.

Griffiths PD, Clark DA, Emery VC. ß-herpesviruses in transplant recipients. J Antimicrob Chemother. 2000;(45):29-43.

The three betaherpesviruses known to infect humans are cytomegalovirus (CMV) and human herpesviruses 6 and 7 (HHV-6 and -7). All three viruses can infect opportunistically after organ transplantation. CMV causes a variety of end-organ diseases, including pneumonitis, hepatitis and gastrointestinal ulceration. Patients who develop overt CMV diseases have significantly higher CMV viral loads than infected patients without evidence of clinical disease. A high CMV viral load largely explains the previously described risk factors for the development of CMV disease, which include donor/recipient serostatus before transplant and viremia after transplant. CMV also causes some cases of allograft rejection, which can be prevented by antiviral prophylaxis. Application of similar quantitative methods for the study of HHV-6 and -7 have shown that HHV-6 and CMV are significantly and independently associated with biopsy-proven graft rejection after liver transplantation. The full clinicopathological significance of the betaherpesviruses may, thus, be greater than is currently appreciated.

Simmons A. Clinical manifestations and treatment considerations of herpes simplex virus infection. J Infect Dis. 2002;186(suppl 1):S71-S77.

Herpes simplex virus (HSV) types 1 and 2 cause infections manifesting as dermatologic, immunologic, and neurologic disorders. Some of the most important manifestations and complications of HSV infection are considered here in a neuroanatomic context. This discussion should aid in understanding the pathogenesis and, in some cases, diagnosis and management of associated HSV-related diseases. The sensory nervous system, rather than skin and mucous membranes, is the primary target of HSV infection. With the intention of extending the benefits of acyclovir, valacyclovir is now being explored in a number of HSV-related conditions. This review extends contemporary thinking about how new antiherpetic drugs might be put to greater therapeutic use in the future.

Tang YW, Mitchell PS, Espy MJ, et al. Molecular diagnosis of herpes simplex virus infections in the central nervous system. J Clin Microbiol. 1999;(37):2127-2136.

Herpes simplex virus (HSV) causes a wide spectrum of clinical manifestations in the central nervous system (CNS) of infants (encephalitis with or without disseminated visceral infection) and adults; the virus likely accounts for at least 10 to 20% of all viral encephalitis in the United States. Effective antivirals-acyclovir (9-[hydrorulteoxymethyl]guanine), vidarabine, and as of recently the prodrugs valacyclovir (converted to acyclovir) and famciclovir (converted to penciclovir)-are available for therapeutic intervention. Rapid laboratory diagnosis is now essential for timely treatment of CNS disease caused by HSV, and as is the case for human immunodeficiency virus (HIV), the use of molecular diagnostics testing in this setting has become popular because of the availability of a specific and effective antiviral therapy. In this mini-review, we describe the technical aspects of the test required for successful implementation for routine diagnostic testing, the extended information now made available by molecular analysis, and the expanded clinical spectrum of disease now recognized by use of this assay.

Whitley RJ, Roizman B. Herpes simplex virus infections. Lancet. 2001;(357):1513-1518.

Herpes simplex virus (HSV) is a member of the herpesviridae family. Recognized since ancient Greek times, the virus frequently infects human beings, causing a range of diseases from mild uncomplicated mucocutaneous infection to those that are life threatening. In the past 50 years, substantial advances in our knowledge of the molecular biology of HSV have led to insights into disease pathogenesis and management. This review provides a contemporary interpretation of the biological properties, function, epidemiology, and treatment of HSV diseases.