Sensitive, qualitative detection of human herpesvirus-6 and simultaneous differentiation of variants A and B.
BACKGROUND
The current limitations of laboratory testing for the detection of human herpesvirus virus 6 (HHV-6) in clinical specimens with low HHV-6 viral loads make this area a priority for further research and development.


OBJECTIVE
To develop and validate a sensitive qualitative assay for simultaneous HHV-6 detection and variant differentiation.


METHODS
We developed a diagnostic procedure, which combines a magnetic bead-based nucleic acid extraction, PCR amplification, and colorimetric microtiter plate identification (MAG-PCR-EIA), for the sensitive detection of HHV-6 and the simultaneous differentiation of HHV-6A and HHV-6B.


RESULTS
Analytic sensitivities of the MAG-PCR-EIA assay were 10 copies per reaction for both HHV-6A and HHV-6B variants, which is equivalent to 20 copies/ml when 1ml of clinical specimen was processed. A proficiency panel containing 11 blinded specimens covering HHV-6A viral loads from 0 to 100,000 copies was tested, and the MAG-PCR-EIA was able to detect the lowest concentration at one copy in 200microl. A panel of 27 urine specimens, which were collected from patients with and without chronic fatigue syndrome, was tested by the MAG-PCR-EIA. HHV-6 was detected in two (HHV-6A) patients who have chromosomally integrated HHV-6A and in one (HHV-6B) patient who was a healthy control and diagnosed as cervical cancer later on. The HHV-6 results did not correlate with results previously determined by HHV-6 antigenemia in urine.


CONCLUSIONS
With large specimen volumes processed and an additional signal amplification incorporated, the MAG-PCR-EIA provides a sensitive and qualitative system for HHV-6 detection and simultaneous variant differentiation. Clinical relevance of the assay awaits further investigation.
Li
Haijing
H
Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.
Meng
Shufang
S
Levine
Susan M
SM
Stratton
Charles W
CW
Tang
Yi-Wei
YW
D003124:Colorimetry / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D015654:Herpesvirus 6, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019349:Roseolovirus Infections / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D014556:Urine / Q000821:virology
D004279:DNA, Viral