CASE FILES: BIOCHEMISTRY
It is applicable to a wide variety of clinical, pathologic, or forensic specimens,
as well as to formalin-fixed tissue, inactivated bacterial cultures, and archaeo-
logical specimens. However, since PCR detects nucleic acids from both liv-
ing and dead microbes, this must be taken into account if PCR is used to
monitor response to therapy.
Prior to the PCR method for detection of HSV-1 and HSV-2, clinical spec-
imens were analyzed using immunologic methods to confirm the virus iden-
tity following a viral culture. Such methods are time-consuming (3 to 12 days)
and expensive. In addition, cultivation of the virus is not always successful.
Turnaround time for viral culture averaged 108 hours for positive results and
154 hours for negative results. In comparison, the present PCR method of
HSV detection is specific, relatively fast and accurate. The method allows
detection of one to ten virus particles in the presence of microgram quantities
of cellular or heterogeneous DNA. The PCR assay offers increased sensitivity,
specificity, and improved turnaround time (24 to 48 hours) when compared
with traditional viral culture techniques. These methods have allowed a better
understanding of the physiopathogeny of the disease. In particular, PCR has
revealed the importance of asymptomatic viral shedding in infected patients.
PCR also helps diagnosis in many situations where viral isolation by culture
proves difficult or impossible, for example, in treated or atypical lesions, or in
newborn central nervous system infections. PCR has demonstrated the exis-
tence of prolonged viremia in infected newborns. PCR helps sequencing of the
viral genome for further epidemiologic studies or analysis of resistance to
antiviral drugs. Recently, PCR-derived techniques have been developed to
quantify viral load in real time, thus allowing a diagnosis in a few hours.
After amplification of the samples using PCR the identification of the
virus species can be achieved through restriction enzyme digestion, which
yields a unique pattern of different fragment sizes characteristic for each her-
pesvirus (or other pathogen). Restriction endonucleases are enzymes able to
cut double-stranded DNA at specific palindromic recognition sequences
that are mostly four to six nucleotides long. Furthermore, a well-designed
restriction enzyme panel allows the discrimination between human herpes virus
6 variant A and variant B. In this way, this method can readily detect human
herpesviruses, including occasional multiple infections, in a variety of clinical
samples. When PCR assay was compared to isolation and electron microscopy
for the detection of HSV in clinical samples, all specimens positive by con-
ventional methods were also positive by PCR. However, in a number of clini-
cal specimens in which HSV could not be detected by conventional methods,
PCR was able to demonstrate the presence of the virus.
Treatment of HSV typically involves the use of nucleoside analogs such as
acyclovir (acycloguanosine) that inhibit the viral DNA polymerase. Acyclovir
is a prodrug that is activated by viral thymidine kinase. The activated drug,
acyclo-GTP, inhibits the viral DNA polymerase by acting as a chain termina-
tor when it is incorporated into viral DNA.