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JOURNAL OF CLINICAL MICROBIOLOGY, July 1999, p. 2127–2136 0095-1137/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 37, No. 7

MINIREVIEW Molecular Diagnosis of Herpes Simplex Virus Infections in the Central Nervous System YI-WEI TANG,† P. SHAWN MITCHELL, MARK J. ESPY, THOMAS F. SMITH,*

AND

DAVID H. PERSING

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905 tests lack the sensitivity and specificity required for definitive diagnosis of HSV CNS infection. PCR amplification of HSV DNA in CSF specimens is now the recognized reference standard assay for the sensitive and specific diagnosis of CNS infections caused by HSV. Nine years ago, two seminal publications appeared in the same journal issue (80, 84); these reports predicted the utility of this technology for the laboratory diagnosis of HSV CNS infections. The initial report described an 18-year-old woman who presented with a 1-week history of headache, fever, mild confusion, and severe pseudobulbar palsy. HSV DNA was amplified in the CSF of this patient by PCR; serologic tests (seroconversion by complement fixation) and electroimaging tests (electroencephalography and computerized tomography) supported the diagnosis (80). In a second publication, Rowley and colleagues detected HSV DNA from the CSF of four patients with HSV encephalitis documented by recovery of HSV from brain tissue; in contrast, CSF from six patients with other microbial causes of encephalitis or systemic lupus erythematosis gave negative results (84). Subsequently, verification of these findings was carried out in several laboratories (3, 4, 8, 20, 27, 30, 45, 56, 68). In this minireview, 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.

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 (60). Effective antivirals—acyclovir (9-[2-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 (106), and as is the case for human immunodeficiency virus (HIV), the use of molecular diagnostic testing in this setting has become popular because of the availability of a specific and effective antiviral therapy. Conventional laboratory diagnosis has traditionally depended on cell culture recovery of the virus from brain tissue or cerebrospinal fluid (CSF), detection of virus-specific intrathecal antigen or antibodies, or imaging techniques that provide characteristic wave patterns or focal inflammatory loci in the temporal lobes of the cerebral cortex. In general, these assays have not been adequate for the routine laboratory diagnosis of CNS disease caused by HSV (Table 1). Recovery of HSV from brain tissue has been considered the “gold standard” laboratory test for diagnosis of this CNS disease; however, despite claimed performance characteristics of 100% for both sensitivity and specificity, the invasive surgical procedure is controversial and rarely used for this diagnostic purpose (34, 42, 106). In addition, laboratory experience has documented the rare recovery of HSV from CSF specimens. For example, of 425 viral isolates recovered from this source at the Mayo Clinic over a 12-year period (1984 to 1996), only 9 were HSV (2%). Serologic diagnosis of HSV CNS infections by detection of intrathecal antibody is of little clinical value since the immune response is detected in only a few patients early in the course of HSV CNS disease and in most patients only after 2 to 3 weeks (31, 92, 105). Serologic tests also need to be standardized by concomitant measurement of albumin to determine that virus-specific antibody has not passively diffused from serum into the CSF, thereby yielding false-positive results (55, 63, 69). Finally, computerized tomography, electroencephalography, and magnetic resonance imaging techniques can provide helpful clinical direction, but results from these

CNS DISEASE CAUSED BY HSV Clinical features. HSV infections of the brain can be subdivided into three categories: neonatal HSV infections, which usually are caused by HSV type 2 (HSV-2); HSV encephalitis, most commonly caused by HSV-1; and recurrent aseptic meningitis (Mollaret’s meningitis), which is mainly associated with HSV-2. Neonatal HSV infection, the most serious consequence of genital HSV infection, occurs at an incidence of 1:3,500 to 1:5,000 births in the United States. Most neonatal infections result from the retrograde spread of HSV-2 secondary to maternal genital infection or via passage of the infant through an infected maternal genital tract. Neonates may present with infection localized to the skin, eyes, and mucosa or to the CNS, or they may present with a disseminated infection. The mortality rate for untreated infants who develop disseminated infection exceeds 70% (106). Early institution of therapy can substantially reduce the morbidity and mortality of mucocutaneous infections and the mortality rates of disseminated and CNS infections (105). The diagnosis of neonatal HSV infection is straightforward if skin vesicles are present. However, in many cases, these pathognomonic lesions are ab-

* Corresponding author. Mailing address: Department of Laboratory Medicine and Pathology, Hilton 470, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Phone: (507) 284-2102. Fax: (507) 2844272. E-mail: [email protected]. † Present address: Departments of Medicine and Pathology, Vanderbilt University Medical Center, Nashville, TN 37232-2605. 2127

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J. CLIN. MICROBIOL. TABLE 1. Laboratory techniques for specific diagnosis of HSV infection in the CNS

Test

Ease of performancea

Antigen detection

1

Cell culture

Serology PCR

2–3

2 3–4

Turnaround time

1–3 h 2–7 days

4–6 h 1–2 days

Result interpretation

May indicate infection if correlated with symptoms Indicates active infection

Indirect; probably indicates active infection Indicates active infection

Advantage(s)

Disadvantage(s)

Rapid

Poor sensitivity and specificity

Isolate available for phenotypic antiviral susceptibility testing Potential for automation

Very poor sensitivity; timing of early specimen collection critical

High sensitivity and specificity

Facility requirement; false positive due to carryover contamination and false negative due to inhibitors in specimen

Results generally retrospective

a Performance scores: 1, could be performed in most routine clinical laboratories; 2, could be performed in reference clinical laboratories; 3, could be performed in specialized research laboratories; 4, could be performed in laboratories with skilled technologists and space and equipment dedicated to performing molecular techniques.

sent, which presents diagnostic difficulties regarding distinguishing HSV infection from similar syndromes caused by rubella, cytomegalovirus (CMV), or toxoplasma. Neonatal herpes and the associated problems of diagnosis and perinatal transmission have been discussed at length elsewhere (106). HSV remains the most common cause of severe sporadic fatal encephalitis. In adults, this necrotizing encephalitis involves the medial temporal and inferior frontal lobes; recent reports indicate that levels of cytokines and other markers of immune activation in CSF are elevated (6, 7). Early diagnosis of CNS disease due to HSV is important for initiation of antiviral therapy and effective clinical response. HSV can be cultured from the CSF in only about 4% of cases (69, 92). Brain biopsy via an open craniotomy can increase the diagnostic efficiency for recovery of HSV in cell cultures (about 45%), and the procedure provides tissue specimens for laboratory diagnosis of other treatable conditions in approximately 7 to 16% of cases. However, serious complications, including hemorrhage, may occur in 2% or more of patients, and falsenegative results may occur in approximately 4% of patients, probably because of the focal nature of the disease. Mollaret’s meningitis is a generally mild form of recurrent aseptic meningitis. Episodes last a few days and may recur over a period of months or years (98, 108). Mollaret’s meningitis has been recognized in sexually active women who experience reactivation of latent virus in the presence or absence of clinically apparent genital infection a few days prior to onset of CNS symptomalology (89, 98). CSF analysis indicates a predominantly lymphocytic pleocytosis of 200 or more cells per ml. Large endothelial (Mollaret) cells, likely monocytes, may be present in the CSF. Pathogenesis. The pathogenesis of herpesvirus-associated encephalitis remains poorly understood. Limited studies with animal models indicate that both the olfactory and trigeminal tracts can provide a neurological avenue for HSV to reach the CNS (48, 90). HSV has a marked tropism of cells of the peripheral nervous system and CNS (39). Several studies have revealed that the molecular basis for HSV neurotropism may include the presence of inverted repeats of the genome and structural features of the thymidine kinase (TK) gene; these

sequences may convey enhanced neurovirulence (16, 18, 46). Recently, in studies of gene function, one gene that maps to the inverted repeats of the unique long segment of HSV DNA, the g134.5 gene, appeared to significantly modulate neurovirulence. When both copies are deleted or a stop codon is inserted into the carboxyl terminus of the gene, neurovirulence is ablated (18). Host response to infection may also play a pathogenetic role; interleukin-6 (IL-6) has been suggested to be involved in the pathogenesis of several diseases in humans, including inflammatory and autoimmune disorders as well as lymphoid malignancies (50). In the hyperthermia- and UV light-induced mouse models of HSV infection, treatment with anti-IL-6 antibodies results in a significantly lower frequency of ocular reactivation compared with that in mice treated with a control immunoglobulin (58). HSV has been demonstrated to induce a concomitant release of IL-6, thereby disturbing immune homeostasis (39, 50). Host immunogenetic factors have now been identified that may influence the risk of becoming infected with certain pathogens, as well as the rate of disease progression once infected. For example, resistance to HIV type 1 (HIV-1) infection, both in vitro and in vivo, has been associated with an internal 32-bp deletion in the human CC-chemokine receptor 5 gene (23, 87) and mutations in the promoter region which are associated with increased expression are linked with rapidly progressive disease (67). Mutations in the gamma interferon receptor (IFN-gR) predispose patients to severe disseminated atypical mycobacterial infections (71) and fatal infection with Mycobacterium bovis BCG, the organism most widely used in tuberculosis vaccines (49). Genetic polymorphisms in the IL-12 receptor gene are associated with severe tuberculosis and Salmonella infections (1, 25). Finally, polymorphisms in three codons within exon 1 of the mannose-binding lectin (MBL) genes have been associated with several recurrent infections as well as the persistence of hepatitis B virus infection (99). Taken together, these reports indicate the role of host immunogenetic factors as a determinant of microbial susceptibility and suggest that evaluation of these genetically variable loci may provide diagnostic information relating to clinical severity of infectious diseases. In one of our recent studies, we were able to amplify

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IFN-gR and MBL alleles by PCR amplification of human genomic DNA directly in CSF from patients with Mollaret’s meningitis. While no nonconsensus polymorphisms were found in exons 2 and 3 of the IFN-gR gene, an R52C naturally occurring polymorphism, resulting in an arginine-to-cysteine change at codon 52 of exon 1 of the MBL gene, was overrepresented (93). The pathogenesis of recurrent HSV meningitis remains obscure. As demonstrated by previous reports (9, 77, 98) and our previous study (93), the syndrome occurs almost exclusively as a result of HSV-2 infection. HSV-2 persists in a latent state in sensory ganglia, and reactivation may be associated with genital lesions and episodes of aseptic meningitis. Several studies established that HSV, particularly HSV-2, is a common cause of Mollaret’s meningitis even in the absence of skin or genital lesions (9, 77, 98). In our previous study (93), only 39.1% of patients had previous local lesions. Although patients usually have their own distinct recurrence patterns, high stress and pregnancy have been reported to be associated with the recurrence (9, 108). Mollaret’s meningitis was associated with a strong predominance of young females in our population, perhaps indicating hormonal influence on immune responses to HSV-2 at the level of helper T-cell (Th) responses and Th1/ Th2 cytokine balance (38, 78, 103). Ultimately, it is likely that multiple factors, including viral, host, and other environmental determinants, play a role in the pathogenesis of HSV CNS diseases. Treatment. The first choice of antiviral drugs now available for HSV treatment is acyclovir, because it is relatively nontoxic and easier to administer. Acyclovir has largely replaced other antiviral drugs as the treatment of choice for HSV infections. Acyclovir is phosphorylated by a virus-specific enzyme, TK, to acyclovir monophosphate, which is subsequently phosphorylated to acyclovir triphosphate by cellular kinases. The triphosphate form of acyclovir competes with the natural substrate dGTP for viral DNA polymerase, resulting in termination of DNA synthesis. Treatment of encephalitis with this agent reduces the mortality rate to 19% 6 months after treatment, compared with 50% among those patients treated with vidarabine and .70% among those patients treated with placebo in prior studies (92, 107). The best available therapy for HSV encephalitis is intravenous acyclovir (30 mg/kg of body weight/day), which is given for a period of 14 to 21 days (92, 107). The rationale for the longer course of therapy is the reduction of the probability of relapse. Therapy with intravenous acyclovir reduces the mortality of HSV encephalitis from about 70% to less than 30% and also reduces morbidity (92, 107). PCR Detection of HSV DNA by PCR may be the prototypical application of molecular diagnosis as applied in the routine clinical laboratory. Beyond the realized goal of amplification and molecular sequencing of HSV DNA products, PCR has the potential for rapidly expanding our understanding of neurovirulence, host responses to CNS disease caused by this virus, and correlation of genotypic with phenotypic resistance assays of HSV to antiviral drugs and has the potential for yielding better clinical outcomes and reduced use of acyclovir compared with empiric therapy (36, 62, 93, 97). Prior to the implementation of HSV PCR in our laboratory, we prospectively analyzed 51 consecutive CSF samples (submitted to our laboratory for HSV detection) by amplification of virus-specific DNA by PCR. Twenty CSF specimens from patients without CNS disease were tested as controls. Alto-

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gether, 15 of 21 patients had evidence of CNS infection (5). Three of the 15 specimens contained HSV DNA; clinical review indicated disease consistent with HSV encephalitis or meningitis. The 12 patients infected with other microbial agents and the 56 specimens (20 control and 36 patient specimens) were negative for HSV DNA by PCR (specificity, 100%). A pivotal study for establishing the sensitivity of PCR for laboratory diagnosis of HSV CNS disease was reported by Lakeman and Whitley (60). They detected HSV DNA in 53 of 54 archived CSF specimens from patients enrolled in a collaborative antiviral study and who had biopsy-proven CNS infection with this virus. Actually, sensitivity of the PCR assay probably exceeded that of culture of brain tissue, in that HSV DNA was amplified in the CSF of 3 of 47 (6%) patients whose tissue failed to yield the virus by conventional techniques. Sample transport and processing. CSF (0.5 ml) is collected by lumbar puncture and transported to the laboratory at 4°C in a sterile screw-cap vial. Samples are stable for extended periods at 4°C (days to weeks); however, if the specimen is received frozen, multiple freeze-thaw procedures should be avoided. Specimens should be shipped, received, and processed in a manner that will not cause cross contamination of samples; i.e., dedicated accessioning and aliquoting procedures should be established. Several extraction methods are commercially available or have been described for extracting viral nucleic acids for amplification procedures (Table 2). Ideally, an extraction procedure should isolate, concentrate, and provide a pure product free of inhibitors; such materials may reduce the efficiency of amplification and ultimately lower the sensitivity of the PCR. Several factors must be weighed in the selection of an appropriate extraction method: sample type, sample volume, desired sensitivity, need for multiple tests to be performed with one sample, toxicity of reagents, efficiency of removal of PCR inhibitors, time of extraction, and special equipment needs. In our laboratory, CSF samples (0.2 ml) for detection of HSV DNA by PCR are processed with a commercially available kit (IsoQuick; ORCA Research, Inc., Bothell, Wash.) according to the manufacturer’s instruction with one modification: the addition of 20 mg of Pellet Paint coprecipitant (Novagen, Madison, Wis.) to each specimen as a carrier during isopropanol precipitation. IsoQuick utilizes the chaotropic properties of guanidine isothiocyanate, which disrupts cellular integrity and inhibits nucleases; the procedure is designed to extract both DNA and RNA in yields comparable to that obtained by the phenol-chloroform method but without the use of hazardous chemicals. The IsoQuick extraction method detects as few as 1 to 10 copies of plasmid HSV DNA extracted from 50 ml of CSF. A comparative study has indicated that extraction of DNA from specimens with acid guanidinium thiocyanate was found to be superior to other methods, including extraction with Proteinase K lysis and phenol-chloroform, proteinase K and sodium dodecyl sulfate, RNasin and sodium dodecyl sulfate, and LiCl plus 6 M urea (13). Application variables: controls, target, and genotype. Seven major loci, distributed throughout the unique long (UL) (5) and unique short (US) (2) regions of the 152-kb genome of HSV, have been reported for the detection of viral DNA in clinical specimens (Fig. 1). All genetic targets but one (TK; UL23) code for structural components of the virion; nucleotides in these genes are conserved relative to TK (Table 3). Differentiation of the two types of HSV is often useful for epidemiologic purposes. For example, HSV-1 has been almost exclusively associated with focal, necrotizing forms of encephalitis typically localized to the temporal lobes of the brain. In

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MINIREVIEW TABLE 2. Examples of commercially available kits for nucleic acid extraction in CSF specimens Reagent

Principle

Comment(s)

IsoQuick (ORCA Research, Inc., Bothell, Wash.) DNAzol (Life Technologies GibcoBRL, Grand Island, N.Y.) QIAamp (QIAGEN, Inc., Santa Clara, Calif.)

Chaotropic lysis

RapidPrep (Pharmacia Biotech, Piscataway, N.J.) Micromix (Tri-Delta Diagnostics, Cedar Knolls, N.J.) GNOME (Bio 101, Vista, Calif.) Dynabeads DNA DIRECT (DYNAL, Lake Success, N.Y.) NucliSens (Organon-Teknika Corp., Durham, N.C.) XTRAX (Gull Laboratories, Inc., Salt Lake City, Utah) InstaGene Matrix (Bio-Rad, Hercules, Calif.)

Spin column

May also be used for RNA extraction; extraction times are 90 min to 3 h; properties of organic phase may inhibit amplification 10- to 30-min protocol; 70–100% recovery rate; application to paraffin tissue samples Available for several specimen types; free of inhibitors; 96-well capability enhances high-volume testing; DNA ranges up to 50 kb; extraction times are 20 to 120 min Not applicable to high volume testing

Spin column

2 to 6 mg of pure DNA in 10–60 min

Chaotropic lysis Magnetic separation

Three-step, 60- to 90-min protocol Precipitation not required

Silicon binding

For both DNA and RNA extraction; relatively quick protocol; applicable to large-volume specimens One sample completed in 3.5 min; procedure requires a microwave; successful for stool samples

Chaotropic lysis Spin column

Chaotropic lysis Chaotropic lysis

contrast, HSV-2 generally causes meningitis and milder forms of CNS disease. PCR of HSV DNA in CSF specimens has not yielded technically convenient methods for HSV genotype designation. Thus, because of the lack of variability in the nucleotide sequence of the HSV genes coding for structural components of the virus, routine differentiation of the two genotypes has not been routinely obtained. Molecular methods for genotype distinction have included unique enzyme restriction sites, nested amplification using type-specific primers, multiplex PCR, type-specific probes, direct sequence analysis, and allele-specific PCR (14, 15, 53, 60). These techniques can be technically challenging. For example, in our experience, only a single restriction enzyme (KspI) could be used to differentiate a 290-bp amplicon generated by both HSV genotypes (68). Further, recognition of HSV-2 strains lacked unique enzyme restriction sites for specific identification, and

these amplicons could only be inferred by the lack of a cleavage site. In contrast to the other major loci of the HSV gene which have been used as targets for PCR of CSF specimens, the TK gene has a highly variable nucleotide sequence (Table 1). Analysis of a 335-bp amplicon within the TK gene has revealed 51 nucleotide positions that are consistently present for differentiation between HSV-1 and -2. Of 20 HSV strains identified by serotyping in our laboratory, all were specifically matched according to genotype results obtained by using capture probes in an enzyme immunoassay detection format (see below). Similarly, nucleotide sequence analysis of over 200 HSV strains from genital and dermal sites completely agreed with serotype determinations using specific monoclonal antibodies. Interestingly, the first few months of routine genotypic analysis of HSV DNA amplicon directly from CSF specimens from

FIG. 1. Representation of the HSV genome. The whole HSV genome is approximately 152 kbp. The relative locations of several important genes are shown. Abbreviations: TRL and TRS, long and short inverted repeats, respectively; IRL and IRS, long and short inverted repeats, respectively.

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TABLE 3. Target loci in the HSV genome for detection of DNA in CSF specimens HSV gene or transcriptional unit

Designation of protein

Amplicon size (bp)

Reference(s)

UL23

TK

351 335 110

27, 53 68, 94 72

UL27

Glycoprotein B

136 149

79 60

UL30

DNA polymerase

92 330 518 476 298 294 546; 549 229; 241 290 163 330

4, 5, 12, 37, 41, 51, 84 3, 45, 52, 53 85 100 70 101 14 56 68 83 35

UL42

DNA binding

159; 225 277

15 81

UL44

Glycoprotein C

840

91

US4

Glycoprotein G

162 187

8, 15 20

US6

Glycoprotein D

221 141 200; 297 421

15, 20, 22, 80 2, 102 47, 82 59

92 patients (male, 35; female, 57) has revealed a predominance of type 2 (n 5 56; 61%) compared with type 1 (n 5 36; 39%) strains of the virus. This trend suggests a proactive trend by clinicians for collection and submission of specimens from patients with primary or recurrent (e.g., Mollaret’s) meningitis. Laboratory design and control of carryover contamination. Carryover contamination can be controlled by the addition of specific modifications to amplification protocols (33, 76, 86); these protocols are usually adequate to control PCR product contamination in laboratories with low to medium volume. Postamplification photochemical inactivation of amplified products has been described with the use of isopsoralen. This chemical is part of the PCR master mix but is not involved in the amplification process. At a low concentration (;25 to 50 mg/ml), isopsoralen may be incorporated into the PCR master mix solution and target nucleic acid. After amplification, and before the PCR tube is opened, the PCR tube is exposed to light at 300 to 400 nm. Nucleic acid polymers are covalently bound, preventing further amplification of the molecule (33, 44, 86). Preamplification inactivation of dUTP containing PCR products by uracil-N-glycosylase has become routine in many laboratories. PCR products are produced that contain deoxyuracil instead of TTP. If dUTP-containing products are present as contaminants, uracil-N-glycosylase will selectively recognize and cleave uracil residues from the DNA backbone, preventing further amplification of the molecule (65, 66, 86). For high-volume laboratories, in which large numbers of positive results are generated on a regular basis, additional precautions may be warranted, especially since no commercial

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kits yet exist for PCR detection of HSV. Recommendations include a minimum of three rooms dedicated for reagent preparation, sample extraction, and target loading and a PCR product analysis area (76). Each room should have dedicated laboratory coats or gowns, gloves, foot covers, and cleaning supplies. Individual pipettes, racks, biological safety cabinets, workstations, refrigerators, and freezers should be labeled specifically for specimen or reagent usage. Barrier pipette tips and/or positive displacement pipettes minimize nucleic acid contamination between specimens. Reagents for preparation of a master mix should be aliquoted for individual or unit use. Importantly, each day, personnel should work only in a direction from a PCR target- or product-free room to the amplicon analysis area. Random access of personnel to all PCR areas will inevitably lead to target or product contamination and nonspecific test results. The liberal use of UV light in PCR facilities, such as in biological safety cabinets, and surface treatment with 10% bleach also reduce the likelihood of contamination events (19). Assay controls. Proper use of assay controls is an integral part of any PCR protocol. Positive, negative, and reagent blank (no target) controls monitor extraction, amplification, and analysis steps as a means of troubleshooting PCR performance. For example, CSF specimens may contain inhibitors (hemolyzed or anthochromic specimens) that interfere with a specimen with HSV DNA with a frequency of 1.2% (60), 1.6% (68), 3.1% (83), and 5.4% (30). Direct assessment of PCR efficiency in CSF samples can be approached by constructing a competitor target designed for amplification by the same primers as native HSV DNA and incorporated into each specimen. Failure to achieve positive amplification of the competitor indicates inhibition of the PCR reaction and a possible falsenegative result. Because both target HSV DNA and the competitor construct compete in the PCR, the sensitivity of the assay may be compromised unless specific care is exercised to avoid such problems. Prospective detection of HSV DNA in CSF specimens. At the Mayo Clinic, from August 1993 through December 1997, HSV DNA was detected in 409 of 6,607 (6.2%) specimens (Table 4). In an interim analysis of 139 CSF samples positive for HSV DNA for which the patient gender was known, 74 (53%) were male (reference 68). However, the proportion of males to females positive for HSV DNA has decreased, beginning in 1995; overall, 67% of 409 positive results in the present study are from women. Concomitantly, laboratory practice at the Mayo Clinic has indicated a trend toward an increasing predominance of specimens from female patients being submitted for HSV testing in the period from 1995 to 1997. Whether this is due to increased recognition of HSV disease or a true increase in the prevalence of HSV among young females is not known at present. HSV quantitation in CSF. Revello and colleagues found that initial DNA levels were not significantly different in patients with encephalitis due to other primary or recurrent HSV infection (83). Viral titers were not related to clinical symptoms, were not predictive of clinical outcomes, and did not decline in either acyclovir-treated or untreated patients. Ando et al. found no correlation between the copy number of HSV DNA in CSF and clinical outcome, but their study indicated that a quantitative PCR assay may be applicable for monitoring response to antiviral therapy (3). Interestingly, levels of HSV may actually increase over the first 5 days of antiviral therapy, perhaps due to the generation of many small fragments of viral genomic DNA when the virus replicates in the presence of acyclovir (81). Detectable levels are often present in the CSF for at least 5 to 14 days and may persist for up to 30 days after

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TABLE 4. Detection of HSV DNA from CSF specimens collected at the Mayo Clinic from August 1993 through December 1997 Yr

1993 1994 1995 1996 1997 Total

No. of subjects positive

No. of specimens tested

No. of positive specimens

% Positive

80 475 1,019 1,951 3,082

3 28 90 122 166

6,607

409

HSV-positive male/female ratio

Male

Female

Unknown gender

3.8 5.9 8.8 6.3 5.4

1 12 37 45 63

1 12 48 74 99

1 4 5 3 4

1.0 1.0 0.77 0.61 0.64

6.2

158

234

17

0.67

the onset of symptoms (2, 8, 60). Using a competitive PCR, Domingues and colleagues were able to quantitate HSV DNA, which ranged from ,25 to 18,000 copies/ml in CSF obtained before or within 4 days of the initiation of acyclovir therapy. Patients with .100 copies of HSV DNA/ml were older, had brain lesions detected by computed tomography, and had poorer outcomes than patients with ,100 copies of the viral genome. In the future, as quantitative protocols and automated assays become available, this assay may be helpful in establishing the prognosis for and in the monitoring of patients with HSV CNS disease (28). Amplicon detection and characterization. The standard means of analyzing PCR products is agarose gel electrophoresis with or without Southern blotting and DNA probe hybridization (5, 54). Interpretation of a stained gel gives a visual representation of expected control results and general assessment of the quality of the PCR. This method should not be viewed as an endpoint, since additional sensitivity can be achieved by incorporation of hybridization techniques, such as Southern blotting. However, because Southern blotting requires various manipulations of the agarose gel and results may take 24 to 48 h, alternative detection methods have been evaluated. One method gaining popularity is colorimetric microtiter plate detection. In a microtiter plate, hybridization and detection of PCR products can be performed using an enzymelinked immunosorbent assay (ELISA) procedure with color formation read on a spectrophotometer (plate reader). Numerous PCR tests have been developed or modified to this format. Some test kits are agent specific; others allow generic detection of amplified products. This technology in combination with rapid extraction and thermal cycling profiles allows same-day test results. We currently use a PCR ELISA system (Roche Diagnostics, Indianapolis, Ind.) in which denatured amplification products are mixed with a hybridization solution containing biotin-labeled DNA capture probe specific for the microorganism of interest (94). The probe hybridizes to the corresponding target DNA sequence if present, and the resulting complexes are captured on streptavidin-coated microtiter plate wells. Specific DNA complexes are detected by conjugate and substrate addition. Color is allowed to develop, and the absorbency is measured (Fig. 2). By designing species-specific biotin-labeled probes within our HSV sequence of interest, we are able to identify HSV-1 and/or HSV-2 strains directly from CSF. Several microtiter plate systems—including but not limited to PrimeCapture from ViroMed Laboratories, Inc., QuantiPATH from CPG, Inc., and GEN-ETI-K from DiaSorin Corp—are commercially available for HSV detection and amplicon identification (65, 94, 104). Mixed infections. Viral coinfection with HSV in the CNS in immunocompetent patients, as determined by PCR, has been

documented. We have demonstrated reactions of HSV-amplified DNA with both type-specific probes in patients with clinical diagnosis of viral encephalitis, suggesting occasional mixed infections with the two genotypes (92), and methods using broad-range PCR for detection of herpesviruses have also documented coinfection. HSV and Epstein-Barr virus (EBV) were detected by PCR in an 11-year-old boy with a clinical course typical for infectious mononucleosis. After several weeks, he developed clinical features of HSV encephalitis (61). In one of our recent studies, of 30 CSF specimens selected on the basis of HSV DNA positivity, 2 were concomitantly positive for human herpesvirus 6 (HHV-6) and 1 was positive for EBV. In

FIG. 2. Detection of digoxigenin-labeled PCR amplicon with the Roche PCR ELISA (digoxigenin detection) kit. (A) During PCR amplification, Taq DNA polymerase incorporates digoxigenin-11-dUTP into the target DNA. (B) A biotin-labeled oligonucleotide probe captures the digoxigenin-labeled PCR amplicon. (C) The probe-amplicon hybrid is immobilized on a streptavidin-coated microtiter plate. (D) The immobilized probe-amplicon hybrid is detected with peroxidase-conjugated anti-digoxigenin antibody and ABTS [2,29-azinobis(3-ethylbenzthiazolinesulfonic acid)] colorimetric substrate.

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the three specimens positive for both herpesviruses, amplicons generated with virus-specific primer sets hybridized specifically to the corresponding virus-specific probe. Sequence analysis of the two amplified DNA fragments demonstrated that they were derived from distinct herpesviruses. These data indicated the presence of DNA specific for two distinct herpesviruses in the same CSF specimen, providing additional molecular evidence that herpesvirus coinfection may occur in the CNS (96). Other herpesviruses, including HSV, CMV, EBV, and HHV-6, have been demonstrated to induce a concomitant release of IL-6, thereby disturbing immune homeostasis (39, 50). This effect may itself be immunosuppressive, which could allow establishment or reactivation of other infectious agents within the host, thereby enhancing the occurrence of coinfection in the CNS. An important question for the future is whether herpesvirus coinfection in the CNS influences the clinical features and disease course of the patient. One patient for whom both EBV and HSV-1 DNA were detected in a CSF specimen experienced a protracted recovery (61). In our previous study described above, among 30 patients for whom HSV DNA was detected in their CSF specimens, 22 were clinically diagnosed with viral encephalitis and appropriately treated with acyclovir. Of 19 patients who were infected only with HSV, only 1 died, while 2 of 3 cases of CNS herpesvirus coinfection (HSV plus HHV-6) confirmed by molecular methods were fatal, even though these patients were treated with acyclovir (96). The significance of herpesvirus coinfection in the CNS and associated clinical manifestations requires further investigation. Antiviral resistance. The number of immunocompromised patients has increased over the last decade as a consequence of aggressive chemotherapy regimens, expanded organ transplantation, and the rising incidence of HIV infection (36). With this change in disease patterns, some HSV strains have developed resistance to acyclovir as well as other nucleoside analogues (40). Isolates of HSV resistant to acyclovir, which were initially strictly a laboratory phenomenon, have now become a clinical routine entity, especially in immunosuppressed patients (21, 73). Resistant isolates have been recovered, although less frequently in immunocompetent patients with recurrent genital herpes (57). HSV can develop resistance to acyclovir through mutations in the viral gene encoding TK, either through the generation of TK-deficient mutants or through the selection of mutants possessing TK that is unable to phosphorylate acyclovir (36, 75). Clinical isolates resistant to acyclovir are almost uniformly deficient in TK, although isolates with altered DNA polymerase have been recovered from HSV-infected patients. Until recently, drug resistance was considered rare, and resistant isolates were believed to be less pathogenic, until a series of acyclovir-resistant HSV isolates from patients with AIDS were characterized (32). These resistant mutants were all deficient in TK. Although acyclovir-resistant HSV is susceptible to vidarabine and foscarnet in vitro, only foscarnet has been shown to be effective in the treatment of infection due to acyclovirresistant HSV. Acyclovir-resistant HSV isolates have been identified in cases of pneumonia, encephalitis, esophagitis, and mucocutaneous infection in immunocompromised patients. Association between certain mutations in the viral TK gene and the acquisition of acyclovir resistance provides an in vitro estimate of the probability that an infection will respond to therapy in vivo (36, 75). Molecular techniques are beginning to play a role in rapid detection of resistance by amplifying and sequencing the resistance-associated genes. There is considerable interest in the application of molecular methods directly to clinical specimens to provide more rapid identification of

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resistance, resulting in improved patient outcome and reduction in overall costs. However, application of molecular techniques to the problem of drug resistance depends on a definitive understanding of the relationship between genotypic and phenotypic susceptibilities, and the current status of HSV testing does not yet allow these associations to be made. For example, sequence analysis of a 335-bp portion of the TK gene of over 200 HSV strains isolated from many anatomical sites in addition to HSV amplicons from CSF revealed many naturally occurring polymorphisms that were not correlated with phenotypic resistance to acyclovir (62). FUTURE DIRECTIONS Effective use of PCR. PCR performed with CSF specimens is recognized as the new gold standard for HSV and other organism infections in the CNS (5, 8, 52, 68, 84). However, with implementation of these new assays there is an increasing tendency toward costly overuse of tests in the context of screening patients with nonspecific complaints. The cumulative fee for these assays, several of which may be ordered simultaneously, may exceed $1,000, of which a substantial percentage goes directly toward PCR-related royalties. The vast majority of these tests yield negative results, so cost-effective means of screening specimens to qualify them for PCR analysis are worth exploring. Leukocyte counts and total protein levels in CSF are predictive of CNS infections (43, 47, 64). A CSF pleocytosis is present in 97% of HSV encephalitis cases (69). One of our recent studies has demonstrated that among 732 specimens submitted for herpesvirus detection (HSV, CMV, EBV, or/and varicella-zoster virus), positive results were detected in 24 of 523 (4.6%) specimens with either abnormal leukocyte counts or total protein levels. In contrast, none of 209 specimens with normal leukocyte and protein levels were positive for herpesviruses. Furthermore, more specimens in which leukocyte and protein levels were both abnormal had a higher diagnostic yield of herpesviruses compared to samples in which only one of these indicator tests was positive (95). These results suggest that protein and leukocyte levels in CSF should be analyzed prior to PCR testing for herpesviruses. Eliminating PCR tests for CSF samples with normal leukocyte and protein levels would save almost one-third of costs associated with herpesvirus testing without having a significant impact on patient care. Hierarchical testing may also improve utilization of services. Among the PCR tests for which CNS infection offered, HSV is of great importance. HSV PCR is proven to have high sensitivity (68, 84) and is one of the few tests for which the laboratory diagnosis determines the use of a potentially life-saving therapy (92, 107). HSV was also the most frequently detected pathogen in our study, and none of the 13 HSV-positive patients were found to have any other CSF pathogens (95). In clinical practice, we therefore suggest that when HSV PCR is ordered in combination with other CSF PCR tests, the HSV PCR should be performed first. The presence of HSV would essentially exclude the need for further testing except in unusual circumstances. Addition of enterovirus PCR for detection of another relatively common cause of CNS infections might be considered on a seasonal basis. One stone for two birds: multiplex PCR. One disadvantage of molecular diagnostic kits and many user-developed tests is that they are narrow in scope. Current organism-specific nucleic acid detection methods assume that the physician or laboratorian knows which pathogen is causing the disease—an assumption which, if true, makes the test useful only as an expensive confirmation for the clinical diagnosis. Multiple in-

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fections may be a particular problem for nucleic acid-based methods, because coinfection will not be detected unless the laboratory is specifically instructed to look for multiple organisms, e.g., HSV and HHV-6 in CSF (96). In the future, the most useful molecular diagnostic tests will be those that can simultaneously test for more than one organism. Multiplex PCR, which utilizes multiple primers within a single reaction tube to amplify nucleic acid fragments from different organisms, may be used to solve this problem (17). There have been several reports describing the detection of HSV among herpesviruses and other pathogens by multiplex PCR (10, 15, 24, 74). A multiplex, colorimetric microtiter PCR system designed for detecting six different herpesviruses, including HSV-1 and HSV-2, is now commercially available from Argene Inc. (North Massapequa, N.Y.). In general, the multiplex PCR technology is limited by the number of primers which can be included in a single reaction, the primer-primer interference, and nonspecific nucleic acid amplification. However, use of certain polymerases such as TaqGold may facilitate development of multiplex assays that are truly useful in a panel format (11). CONCLUSION The technical and diagnostic impact of PCR for evaluation of HSV CNS disease has been evident in clinical practice. In the past, physicians were limited to assessment of clinical symptomatology of the patient, neurodiadiagnostic imaging information, and culture of brain biopsy specimens for acute diagnosis of HSV CNS disease. Generally, only those with typical and severe CNS infections with this virus were identified; most often, patients were comatose and denied the efficacy of effective antiviral treatment because of the invasive procedures associated with laboratory diagnosis in the early stages of this disease. In contrast, today clinicians can submit CSF specimens rather than a brain biopsy specimen for the detection of HSV DNA by PCR when CNS infection is initially suspected. By this practice, the spectrum of CNS disease caused by this virus has been rapidly expanded to recognition of mild forms and atypical forms of meningitis and encephalitis (26, 29, 30, 88). Accordingly, these findings may promote new management strategies for these forms of HSV CNS disease. In addition, PCR has served to correlate and standardize diagnostic results that can localize temporal abnormalities shown by imaging techniques of computerized tomography, electroencephalograms, or magnetic resonance imaging techniques (30). Besides the new diagnostic dimension and the PCR, this technology provides us the facility for investigating the pathogenesis of HSV CNS disease. Analogous to the findings of the b-chemokine receptor CCR-5 regarding the control of entry of HIV into cells, molecular studies of genes coding for IFN-gR and IL-12 receptor and the MBL and the a134.5 genes can now be expanded to include their role in neuroinvasion by HSV (1, 23, 25, 71, 87, 99). Detection of HSV DNA in CSF is rapid, sensitive, and specific and is clearly the gold standard for the laboratory diagnosis of CNS disease caused by this virus. REFERENCES 1. Altare, F., A. Durandy, D. Lammas, J.-F. Emile, S. Lamhamedi, F. Le Deist, P. Drysdale, E. Jouanguy, R. Doffinger, F. Bernaudin, O. Jappsson, J. A. Gollob, E. Meinl, A. W. Segal, A. Fischer, D. Kumararatne, and J.-L. Casanova. 1998. Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science 280:1432–1435. 2. Anderson, N. E., K. F. Powell, and M. C. Croxson. 1993. A polymerase chain reaction assay of cerebrospinal fluid in patients with suspected herpes simplex encephalitis. J. Neurol. Neurosurg. Psychiatry 56:520–525.

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