Test Id : QHV6C

Container/Tube: Sterile vial

Preferred: Aliquot tube

Specimen Volume: 0.5 mL

Collection Instructions:

1. Collect specimen from collection vial 2.

2. Do not centrifuge.

0.3 mL

As an adjunct in the rapid diagnosis of human herpesvirus-6 infection using cerebrospinal fluid specimens

This test should not be used to screen asymptomatic patients.

Human herpesvirus-6 (HHV-6) is a member of the Herpesviridae family. These DNA viruses contain a capsid surrounded by a lipid envelope. Among members of this group, this virus is most closely related to cytomegalovirus (CMV) and HHV-7. As with other members of the herpesvirus group (herpes simplex virus [HSV]-1, HSV-2, varicella-zoster virus, CMV, Epstein-Barr virus, HHV-7, HHV-8), HHV-6 may cause primary and reactivated infections.(1) Infection with HHV-6 occurs early in childhood. Most adults (80%-90%) have been infected with this virus.

Human herpesvirus-6 was first linked with exanthem subitum (roseola infantum) in 1998; since then, the virus has been associated with central nervous system disease almost exclusively in patients who are immunocompromised.(1) HHV-6 is commonly detected in patients post transplantation. Clinical symptoms associated with this viral infection include febrile illness, pneumonitis, hepatitis, and encephalitis. However, most HHV-6 infections are asymptomatic.(2)

Human herpesvirus-6 is designated as variant A (HHV-6A) or variant B (HHV-6B) depending on restriction enzyme digestion patterns and its reaction with monoclonal antibodies. Generally, variant B has been associated with exanthem subitum, whereas variant A has been found in many immunosuppressed patients.(3) Infection with HHV-6 is very common, approaching 100% seroprevalence in developed countries.(4) In about 1% of the population, HHV-6 can integrate into the host genome. Often asymptomatic in immunocompetent hosts, reactivation can cause serious disease in immunocompromised individuals, particularly those with AIDS and transplant recipients, which can cause rejection of the transplanted organ and even death.(1)

This assay will be used to assist with diagnosis and monitoring of HHV-6 disease in patients who are suspected of having disease due to HHV-6 infection. It will also be used as an initial indicator of infection versus chromosomally-integrated HHV-6.

Undetected

The quantification range of this assay is 500 to 5,000,000 copies/mL (2.70 log to 6.70 log copies/mL)

An "Undetected" test result indicates the absence of human herpesvirus-6 (HHV-6) DNA in cerebrospinal fluid (CSF).

A test result of "<500 copies/mL (<2.70 log copies/mL)" indicates that HHV-6 DNA is detected in the CSF, but the assay cannot accurately quantify the level of HHV-6 DNA.

A test result of ">5,000,000 copies/mL (>6.70 log copies/mL)" indicates that the HHV-6 DNA level present in CSF is above 5,000,000 copies/mL (6.70 log copies/mL), and the assay cannot accurately quantify the level of HHV-6 DNA. A viral load above 5,000,000 copies/mL should raise suspicion for chromosomally-integrated HHV-6 (ci-HHV-6), and additional testing to rule out ci-HHV-6 may be needed.

An "Inconclusive" result indicates that the presence or absence of HHV-6 DNA in the CSF specimen could not be determined with certainty after repeat testing in the laboratory, possibly due to inhibition or the presence of an interfering substance. If clinically indicated, submission of a new specimen for testing is recommended.

The sensitivity of the assay is dependent upon the quality of the specimen submitted.

A negative result does not exclude human herpesvirus-6 virus (HHV-6) infection. Therefore, the results obtained should be used in conjunction with clinical findings to make an accurate diagnosis.

This assay detects nucleic acid and, therefore, cannot distinguish between replicating and nonreplicating virus (ie, remnant viral nucleic acid). Test performance depends on the viral load in the specimen and may not correlate with cell culture performed on the same specimen.

Although this assay may detect dual infections of HHV-6A and HHV-6B, low level infections may be masked when in the presence of a higher concentration of an HHV-6 subtype.

Viral loads may vary between laboratory-developed assays. When monitoring a patient’s HHV-6 viral load, the same method and sample type should be used.

This test cannot be converted to IU/mL. It is recommended to utilize the same test for the duration of the clinical care for consistency in reporting units (copies/mL).

Analytical Sensitivity/Limit of Detection:

Spiked specimens in cerebrospinal fluid (CSF) with human herpesvirus-6 (HHV-6) DNA subtypes A/B (40 for each subtype) for a total of 80 positive specimens were used for establishment of the limit of detection (LOD). The lower LOD of this assay was determined to be 500 copies/mL for HHV-6A and 250 copies/mL for HHV-6B.

Analytical Specificity:

DNA from a panel of 10 organisms from culture collections along with well characterized laboratory strains that cause similar disease or organisms commonly found in CSF were tested to determine if there is any cross-reactivity against the Altona RealStar HHV-6 PCR (polymerase chain reaction) Kit 1.0. No cross-reactivity was observed with the specificity panel.

Analytical specificity was determined by the kit manufacturer (Altona Diagnostics) and did not exhibit cross-reactivity with any of the following targets: BK virus, cytomegalovirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus 1, herpes simplex virus 2, human herpesvirus 7, human herpesvirus 8, human parvovirus B19, JC virus, varicella-zoster virus.

Due to new sequence data, cross reactivity of the HHV6-B detection systems (Cy5/Cy5.5) with some strains of HHV-6A cannot be ruled out. A weak signal in the HHV-6A detection channel (FAM) and HHV-6B (Cy5/Cy5.5) can be observed. This is a known limitation of the assay and will be incorporated in analysis and reporting processes.

Reference Range:

A total of 20 CSF samples collected for noninfectious disease testing (eg, CSF chemistry) were analyzed by the Altona RealStar HHV-6 PCR assay. Of these, 19 were negative. One sample did have a low level of HHV-6 DNA. Given that HHV-6 undergoes latency following primary infection, it is possible to detect viral DNA that may not be associated with active infection or disease. Also, in approximately 1% of patients, HHV-6 may become chromosomally integrated, yielding a positive result by molecular tests.

Although the reference range is typically "negative" for this assay, this assay may detect viral DNA in asymptomatic individuals. However, this assay is only to be used for patients with a clinical history and symptoms consistent with HHV-6 infection and must be interpreted in the context of the clinical picture. This test should not be used to screen asymptomatic patients.

Reportable Range:

The reportable range for this assay is 500 copies/mL to 5,000,000 copies/mL.

1. Agut H, Bonnafous P, Gautheret-Dejean A. Laboratory and clinical aspects of human herpesvirus 6 infections. Clin Microbiol Rev. 2015:28(2):313-335

2. De Bolle L, Naesens L, De Clercq E. Update on human herpesvirus 6 biology, clinical features, and therapy. Clin Microbiol Rev. 2005;18(1):217-245

3. Dockrell DH, Paya CV. Human herpesvirus-6 and -7 in transplantation. Rev Med Virol. 2001;11(1):23-36

4. Campadelli-Fiume G, Mirandola P, Menotti L. Human herpesvirus 6: An emerging pathogen. Emerging Infectious Diseases. 1999;5(3):353-366. doi:10.3201/eid0503.990306

5. Abdel-Haq NM, Asmar BI. Human herpesvirus 6 (HHV6) infection. Indian J Pediatr. 2004;71(1):89-96

6. Dockrell DH, Smith TF, Paya CV. Human herpesvirus 6. Mayo Clin Proc. 1999;74(2):163-170

7  Pellett Madan R, Hand J; AST Infectious Diseases Community of Practice. Human herpesvirus 6, 7, and 8 in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant.2019;33(9):e13518. doi:10.1111/ctr.13518

8. Pawlowska AB, Karras NA, Liu H, et al. Reactivation of human herpesvirus 6 in pediatric allogeneic hematopoietic stem cell transplant recipients. Transpl Infect Dis.  2021;23(1):e13453. doi:10.1111/tid.13453

9. Wang H, Tomatis-Souverbielle C, Everhart K, Oyeniran SJ, Leber AL. Detection of human herpesvirus 6 in pediatric CSF samples: causing disease or incidental distraction? Diagn Microbiol Infect Dis. 2023;107(2):116029. doi:10.1016/j.diagmicrobio

10. Yip CCY, Sridhar S, Cheng AKW, et al. Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar HHV-6 PCR Kit for quantitative detection of human herpesvirus 6. J Virol Methods. 2017;246:112-116. doi:10.1016/j.jviromet.2017.05.001

The RealStar HHV-6 (human herpesvirus-6 virus) PCR (polymerase chain reaction) Kit 1.0 (Altona Diagnostics) and the Exact Diagnostics HHV6-A/B Verification Panels and controls (Bio-Rad) are utilized for this assay. The assay employs TaqMan hydrolysis probe chemistry, with production of visible amplification curves and associated crossing point (Cp) values; no melting temperature curve is produced. A probe specific for HHV-6A DNA is labeled with the fluorophore FAM and is detected on the 465-510 channel. A probe specific for HHV-6B DNA is labeled with the fluorophore Cy5 and is detected on the 533-580 channel. The probe specific for the Internal Control (IC) is labeled with the fluorophore JOE, is detected on the 618-660 channel, and will be used within the mastermix. Using probes linked to distinguishable dyes enables the parallel detection and differentiation of HHV-6A and HHV-6B DNA as well as the detection of the IC in corresponding detector channels of the LC480 II instrument. The MagNA Pure 96 instrument (Roche Diagnostics) using the MP96 DNA and Viral NA Small Volume Kit with an input of 200 mcL and a final elution volume of 100 mcL is utilized for viral nucleic acid extraction. The Pathogen Universal 200 protocol is used.(Package insert: RealStar HHV-6 Virus PCR Kit 1.0. Altona Diagnostics; Version 09/2018)

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This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

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