Diagnostic Microbiology and Infectious Disease 57 (2007) 309 – 313

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Appropriate use of polymerase chain reaction for detection of herpes

simplex virus 2 in cerebrospinal fluid of patients at an inner-city hospital
Nelia Afonsoa, Sunethra Gunasenaa, Karuna Gallaa, Raymond Podzorskib,c,
Pranatharthi Chandrasekara, George Alangadena,4
a
Division of Infectious Diseases, Wayne State University School of Medicine, Detroit, MI 48201, USA
b
Molecular Microbiology Laboratory, Detroit Medical Center University, Detroit, MI 48201, USA
c
Department of Laboratory Medicine, Waukesha Memorial Hospital, Waukesha, WI 53188, USA
Received 8 May 2006; accepted 3 September 2006

Abstract

Polymerase chain reaction (PCR) tests that detect herpes simplex virus (HSV) DNA in cerebrospinal fluid (CSF) are increasingly used to
diagnose central nervous system (CNS) infections caused by HSV. To determine proper utilization of this test at an inner-city hospital, we
performed a case-control study of adult patients, with HSV detected in CSF by PCR. Retrospective review of characteristics of adult patients
hospitalized between 1997 and 2000 with CSF positive for HSV was done and compared to control patients with suspected CNS infection
and negative CSF PCR. CSF from 1174 patients was tested; 20 (1.7%) had HSV DNA detected, 19/20 were HSV-2 and 1 was HSV-1. The
HSV-2 cases were females (74%), with a median age of 41 years, of African-American ethnicity (100%). Of the cases, 90% had acute aseptic
meningitis versus 13% controls ( P b .001). Recurrent meningitis occurred in 42% cases and 3% controls ( P b .001). CSF parameters
significantly associated with HSV-2 positivity was lymphocytic pleocytosis (median leukocyte, 475 cell/mm3, 90% lymphocytes) ( P b .001).
In conclusion, HSV-1 was rarely detected in CSF of patients with suspected CNS infection. HSV-2 is more frequent, predominantly in young
African-American women with lymphocytic aseptic meningitis, and is often recurrent. PCR testing for HSV-2 in CSF at inner-city hospitals
can be greatly reduced by the application of these parameters.
D 2007 Elsevier Inc. All rights reserved.

1. Introduction suggests that these sensitive but expensive PCR tests are
increasingly overutilized (Mitchell et al., 1997). Over a
Herpes simplex virus (HSV) infections of the central 2 1/2-year period, test requests increased from an average of
nervous system (CNS) in adults typically present as 23 to 140 per month without a corresponding increase in the
2 distinct syndromes—viral meningitis caused by herpes rate of detection of HSV-DNA in the CSF (Mitchell et al.,
simplex virus 2 (HSV-2) and encephalitis due to herpes 1997). We wished to assess the use and to provide guidance
simplex virus 1 (HSV-1) (Corey, 2005). for the proper utilization of the PCR test in adult patients
Polymerase chain reaction (PCR) detection of HSV admitted to an inner-city university medical center with
DNA in the cerebrospinal fluid (CSF) is a sensitive and suspected CNS infection. A case-control study was there-
rapid test that has facilitated the diagnosis of HSV ence- fore performed of all patients who had a CSF specimen
phalitis, thus sparing brain biopsies, and has helped identify positive by PCR for HSV-1 or HSV-2 DNA at the Detroit
HSV as a potential cause of aseptic meningitis (Tang et al., Medical Center in downtown Detroit, MI.
1999a, 1999b). However, a report from a referral laboratory

2. Methods
The study was presented in part at the 40th Annual Meeting of the
IDSA, October 24–27, 2002, Chicago (abstract no. 760).
All CSF samples submitted for PCR testing for HSV
4 Corresponding author. Tel.: +1-313-745-7105; fax: +1-313-993-0302. DNA, from adult patients hospitalized between January 1997
E-mail address: galangaden@med.wayne.edu (G. Alangaden). and December 2000 at 3 inner-city urban hospitals within the
0732-8893/$ – see front matter D 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.diagmicrobio.2006.09.002
310 N. Afonso et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 309 – 313

Detroit Medical Center in Detroit, MI, were analyzed. PCR 7.0 (1995; SPSS, Chicago, IL). The 2 groups were
testing was performed at a single core laboratory. The CSF compared using the Student t test and v 2 test for continuous
samples were stored at 4 8C and testing was performed twice and dichotomous variables, respectively. Results are pre-
weekly. Briefly, CSF was extracted and the DNA concen- sented as mean F SD or number and percentage. Statistical
trated using the QIAmp DNA Mini Kit PCR (Qiagen, significance for all tests was established at a nominal P level
Valencia, CA). PCR was performed using oligonucleotide of b.05.
primers specific for a 476-bp DNA signature sequence
present in HSV-1 and HSV-2 DNA polymerase gene. Primer
sequences utilized were (P1) 5V CAG TAC GGC CCC GAC
TTC GTG A 3V and (P2) 5V GTA GAT GGT GCG GGT GAT 3. Results
GTT 3V (Rogers et al 1991). Viral DNA was amplified up to During the 4 years of the study, 1174 CSF samples from
1012-fold by PCR using a 9600 Thermocycler (Applied patients with suspected CNS infections were submitted to
Biosystems, Foster City, CA, USA), and the product was the laboratory with a request for PCR testing for HSV. Of
detected using a colorimetric microtiter-based system with a these, HSV DNA was detected in 20 (1.7%) of the
RNA probe specific for the HSV target (Sharp Signalk specimens; HSV-2 was detected in 19 patients and HSV-1
System, Digene Diagnostics, Gaithersburg, MD, USA) in a single patient. The single patient with HSV-1 DNA in
(Rogers et al., 1991). Amplification was done according to the CSF was an 87-year-old female hospitalized with fever,
the protocol outlined (Roger, 1991) with the following mental status changes, seizures, and was diagnosed as
exceptions: PCR reaction volumes were 50 AL, 10% glycerol having HSV encephalitis.
was added to each reaction, and the PCR was run for 60 Characteristics of the 19 patients with HSV-2 DNA and
cycles (Podzorski et al 2000). Specimens positive for HSV
controls are summarized in Table 1. There were no
by PCR were typed by restriction fragment analysis
significant differences in the demographic characteristics
(Podzorski et al., 2000). Briefly, restriction endonuclease
between cases and controls, the majority being African-
reactions were performed with DrdI or AvaII and were set up
American women, with a median age of about 40 years
according to product inserts (DrdI, New England Biolabs,
(Table 1). Recurrent aseptic meningitis (RAS) was identified
Beverly, MA; AvaII, Roche Diagnostics Corporation, Indian-
in 8 cases (42%) as compared to a single control patient.
apolis, IN) using 25 AL of PCR product directly from the
Five of these patients had a prior history of RAS at the time
amplification reaction and 5 U of each restriction enzyme.
of initial PCR detection of HSV DNA; the other 3 patients
The reactions were incubated overnight. After restriction,
25 AL from each reaction was run on a 3% agarose gel, had a subsequent hospitalization for aseptic meningitis,
stained with ethidium bromide, and visualized using UV which was detected during the follow-up review done at the
illumination (Podzorski et al 2000). No viral cultures were end of 2004. Of these patients, 1 had 5 episodes, 2 each had
performed on the CSF specimens. 4 and 3 episodes, and 3 each had 2 episodes of RAS,
We used the laboratory database to identify adult patients respectively. The single control patient with RAS was a
with a CSF sample positive by PCR for either HSV-1 or 22-year-old woman with 2 hospitalizations for symptoms
HSV-2 DNA. Case records of every patient whose CSF was and CSF findings suggestive of aseptic meningitis; on both
positive for HSV DNA in the CSF were compared to occasions, PCR of the CSF was negative for HSV. No
2 hospitalized control patients. A control was defined as a definite etiology was established. Fever was present in the
patient with suspected CNS infection who had a negative majority of cases and controls (Table 1). However, features
HSV PCR assay of the CSF. Age and gender were selected of meningitis, that is, photophobia, neck pain/rigidity,
for matching controls because most of our HSV-positive headache, and nausea/vomiting, were significantly more
cases had HSV-2 meningitis, which tends to occur common in the HSV-2–positive cases as compared to
predominantly in young women (Read and Kurtz 1999; controls. In contrast, alteration in mental status was
Peter et al., 2001; O’Sullivan et al., 2003). A follow-up significantly more common in controls. Of 19 HSV-
review of the medical records of all patients and controls 2–positive cases, 6 (32%) exhibited all 4 symptoms of
was repeated at the end of December 2004 to determine if meningitis and 9 of the 19 patients (47%) had at least 3 of
any subsequent hospitalizations occurred for CNS infec- the above symptoms. Overall, 17 of the 19 (90%) HSV-
tions, or if any subsequent PCR testing of the CSF was 2–positive cases had clinical features compatible with
performed. The analysis included patient demographics, aseptic meningitis. The 2 patients without symptoms of
clinical presentation, initial and final diagnoses, CSF aseptic meningitis, 1 with colon cancer, and the other with
parameters, radiologic tests, treatment, and outcome. The systemic lupus had symptoms of transverse myelitis. Both
study was conducted in accordance with the policies of the presented with numbness of the trunk and lower extremities.
local human investigation committee. The patient with lupus also had weakness of the left lower
extremity and a vesicular rash consistent with herpes zoster
2.1. Statistical analysis
along the left T9 dermatome. Both patients had evidence of
The data obtained were coded, entered into a data file, transverse myelitis on magnetic resonance imaging (MRI)
and analyzed using SPSS for Windows software, Release scans. No varicella zoster viral DNA was detected by PCR
 N. Afonso et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 309 – 313 311

Table 1 an admitting diagnosis of encephalitis was made in only 1 of

Clinical characteristics and CSF parameters of patients with HSV-2 DNA the controls and in none of the cases.
positive by PCR in CSF and controls with HSV DNA negative in CSF
Seventeen of the 19 cases positive for HSV-2 had
Characteristics at time HSV-2–positive HSV-negative P value complete resolution of symptoms and were discharged
of hospital admission cases (n = 19) (n = 38)
home after a mean hospital stay of 5 days.
African-American 19 (100) 35 (92.1) .529
Eight (42%) of the 19 HSV-2–positive patients received
ethnicity
Underlying medical 12 (63) 33 (87) .85 treatment with acyclovir for a mean of 8 days (range,
comorbidity 5–10 days). The remaining 11 patients who received no
Prior history of 8 (42) 1 (2.6) b .001 antiviral therapy had spontaneous resolution of symptoms
aseptic meningitis and were discharged home before receipt of the positive
Clinical features
PCR results. The mean duration of symptoms of meningitis
Fever 14 (73.7) 23 (60.5) .492
Headache 16 (84.2) 19 (50) .027 in patients treated with acyclovir was 7.2 days (range,
Photophobia 15 (78.9) 5 (13.9) b .001 5–9 days) compared to 4.9 days (range, 3–10 days) in those
Neck pain/rigidity 12 (63.2) 8 (21.1) .004 not treated. Two acyclovir-treated patients and 1 patient in
Nausea/vomiting 10 (52.6) 8 (21.1) .034 the untreated group had a subsequent episode of aseptic
Focal neurologic 2 (10.5) 7 (18.4) .700
meningitis during the follow-up period. Of the 38 control
signs/symptoms
Paraesthesia 1 (5.3) 3 (7.9) 1.00 patients, 3 (8%) received empiric treatment with intravenous
Seizures 2 (10.5) 11 (28.9) .220 acyclovir, which was discontinued in 2 after a negative HSV
Admitting diagnosis of 15 (78.9) 5 (13.1) b .001 PCR was reported. Among controls, an infection was
aseptic meningitis diagnosed in only 14 (38%) patients; the discharge
Admitting diagnosis 0 1 (2.6) .711
diagnoses for controls were non-CNS infection (7 patients),
of encephalitis
CSF parameters stroke (7 patients), seizure disorder (5 patients), viral
Glucose (mg/dL) 65.7 F 23.7 81 F 62.0 .307 meningitis (3 patients), cryptococcal meningitis and HIV
(range, 36–118) (range, 27–351) encephalopathy (2 patients each), CNS tumor (3 patients),
Protein (mg/dL) 92.4 F 32.3 107.7 F 172.7 .706 metabolic encephalopathy (3 patients), and lupus cerebritis
(range, 24–139) (range, 22–953)
(2 patients).
Leukocyte count 475.7 F 317 68.9 F 149.1 b .001
(cell/mm3) (range, 100–1130) (range, 0–600)
Lymphocyte (%) 89 F 21.2 36.9 F 38.7 b .001 4. Discussion
(range, 42–98) (range, 0–100)
Segmented cells (%) 6.3 F 11.2 31.78 F 38.2 .007 In patients with suspected CNS infections hospitalized at
(range, 0–47) (range, 0–99) an inner-city medical center, HSV DNA was detected in
Values are mean F SD or n (%). Percentages are derived from column 1.7 % of 1174 patients who had CSF tested by PCR. Studies
totals.
in the United States and Europe involving children and
adults reported positive rates of 1.2–7.1% (Mitchell et al.,
in the CSF of the patient with herpes zoster. Both were 1997; Jeffery et al., 1997; Read and Kurtz 1999; Tang et al.,
treated with intravenous acyclovir. The symptoms com- 1999a, 1999b; Najioullah et al., 2000; Studahl et al., 2000;
pletely resolved in 1 patient; however, the patient with lupus Cimolai et al., 2001; Minjolle et al., 2002; Sauerbrei and
has residual mild motor weakness in the left lower Wutzler 2002).
extremity. None of the 19 HSV-2 cases had skin lesions Only a single CSF specimen tested positive for HSV-1
consistent with HSV infection at the time of hospitalization. DNA, an observation that differs from earlier studies with a
A computed tomography (CT)/MRI scan of the brain was greater frequency of HSV-1 compared to HSV-2 (Mitchell
performed in 84% of cases and 89% of controls. Radiologic et al., 1997; Jeffery et al., 1997; Tang et al., 1999a, 1999b;
abnormalities were reported in 18 controls (55%) and in Najioullah et al., 2000; Studahl et al., 2000; Cimolai et al.,
3 cases (19%). Among cases, 2 had radiologic features of 2001; Minjolle et al., 2002; Sauerbrei and Wutzler 2002).
myelitis (discussed above) and 1 had features of cerebral The greater frequency of HSV-2 in our study might be due
ischemia. The commonest abnormalities reported among to the inclusion of adult patients only. This is in contrast to
controls were cerebral ischemia or hemorrhage in 8 (44%) reports that included patients of all ages (Mitchell et al.,
and cerebral atrophy in 3 (17%). 1997; Jeffery et al., 1997; Read and Kurtz 1999; Najioullah
The CSF parameters of cases and the controls are listed in et al., 2000; Studahl et al., 2000; Sauerbrei and Wutzler
Table 1. Only a high leukocyte count (median white blood 2002), or only included cases of encephalitis that generally
cell [WBC] count, 476/ mm3) with lymphocytic predomi- caused by HSV-1 in adults (Sauerbrei and Wutzler 2002).
nance (median lymphocyte count, 89%; range, 42–98%) was HSV-1 encephalitis is uncommon and is estimated to occur
significantly associated with HSV-2 positivity. All of the in 2.33 patients per million persons per year (Najioullah
HSV-2–positive cases had abnormal CSF parameters. et al., 2000). Recent studies from North America report a
A diagnosis of aseptic/viral meningitis was recorded at greater frequency of HSV-2 detection compared to HSV-1
admission in 79% of the cases and 13% of controls, whereas (Peter and Sevall, 2001; Simko et al., 2002; O’Sullivan et al.,
312 N. Afonso et al. / Diagnostic Microbiology and Infectious Disease 57 (2007) 309 – 313

2003). Most of HSV-2–positive tests occurred in young The high frequency of CT/MRI brain scans performed in
women as was noted in our study (Read and Kurtz 1999; cases and controls is of interest. Most of our HSV-2 cases
Peter and Sevall, 2001; Studahl et al., 2000; Simko et al., had features of aseptic meningitis without focal neurologic
2002; O’Sullivan et al., 2003). Seroprevalance of HSV-2 has signs. Criteria for appropriate use of CNS imaging in cases
steadily increased with the greatest lifetime cumulative of meningitis include focal neurologic abnormality, abnor-
incidence of 80% in African-American women (Corey, mal mentation, seizure, age N 60 years, immunocompromis-
2005), the group most affected in our study. Therefore, it is ing condition, or a prior CNS disease (Hasbun et al., 2001).
likely that PCR testing will be positive for HSV-2 rather Specific antiviral therapy was administered in only 42%
than HSV-1 if utilized within an inner-city hospital setting. of HSV-2–positive cases. The majority of patients had
This is the first case-control comparison of the clinical spontaneous resolution of symptoms and were discharged
and CSF characteristics of hospitalized patients with HSV-2 before receipt of the PCR results. Both treated and untreated
DNA in the CSF. RAS was significantly associated with cases had subsequent episodes of RAS. This suggests that
HSV-2 positivity. Since the PCR detection of HSV-2 in the despite detection of HSV-2 DNA in the CSF in patients with
CSF of patients with RAS by Tedder et al. (1994), there aseptic meningitis the benefit of specific antiviral therapy
have been additional reports of the association between remains uncertain. Recommendations for antiviral therapy
RAS and HSV-2 (Picard et al., 1993; Jensenius et al., 1998; for RAS are based upon expert opinion and suggest
Kupila et al., 2004). In adult patients with aseptic meningitis treatment should be individualized based upon the number
and HSV-2 DNA in CSF, prior meningitis occurred in 27% and severity of recurrences (Tyler 2004). A randomized
to 67% (Read and Kurtz 1999; Studahl et al., 2000; Simko multicenter evaluation of suppressive therapy with valacy-
et al., 2002; O’Sullivan et al., 2003), similar to the 42% of clovir is ongoing in Sweden (Tyler, 2004).
our cases with RAS. Thus, patients with RAS should be Although our study is retrospective, the findings will
considered for HSV-2 PCR. help identify appropriate patients for CSF testing for HSV-2
The clinical features indicative of HSV-2 positivity were DNA, thereby reducing overutilization of expensive PCR
acute-onset aseptic meningitis (89%) presenting with testing. The cost of a PCR test at our center is $160 with an
photophobia, neck pain/rigidity, headache, or nausea/vomit- estimated cost of approximately $188,000 for the study
ing. HSV-2 infection of the CNS in adults typically presents period. Because only 1.7% of 1174 patients tested had a
as acute aseptic meningitis, generally during primary genital positive CSF HSV PCR, considerable cost saving might
infection (Corey, 2005). Altered mental status was associ- have been realized if the test was applied to selected
ated with a negative predictivity for HSV-2. It is likely that patients. Once the diagnosis of encephalitis versus menin-
PCR testing in many of the controls was requested due to gitis is made, newer real-time PCR assays that specifically
clinical suspicion of encephalitis indicated by fever and identify either HSV-1 or HSV-2 DNA can be utilized (Corey
altered mental status. However, only 1 of the controls had et al., 2005).
encephalitis recorded as the admission diagnosis, and only 3 Thus, CSF specimens from patients with suspected CNS
received empiric acyclovir that was discontinued in 2 after a infections should be stored and PCR testing for HSV-2 be
negative PCR result. performed only in patients especially young women with a)
None of our cases had HSV skin or mucosal lesions. symptoms of RAS or b) patients with acute aseptic
Although skin lesions were noted in 7–18% of patients with meningitis and CSF demonstrating lymphocytic pleocytosis.
CSF positive for HSV-2 (Read and Kurtz, 1999; Simko Application of these parameters would reduce the number of
et al., 2002), a recent study of patients with aseptic PCR tests performed without a proportional decrease in the
meningitis and positive HSV-2 PCR reported no skin positive results for HSV-2 DNA. This would result in cost
lesions at presentation (O’Sullivan et al., 2003). savings without significant impact on clinical outcomes.
Laboratory parameters significantly associated with HSV-2
positivity in our study were elevations in CSF leukocyte
count and lymphocyte predominance. In patients with Acknowledgments
suspected CNS infections, a CSF WBC count of z 5/mm3
We thank Anil Aranha for statistical evaluation of
together with fever, a virus-specific rash, or headaches were
the manuscript.
reported as independent predictors of a positive PCR result
for HSV DNA (Jeffery et al., 1997; Minjolle et al., 2002;
Simko et al., 2002), whereas CSF with normal protein and
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