ORIGINAL ARTICLE Scand J Infect Dis 29: 229-231, 1997

Advantage of Polymerase Chain Reaction in the Diagnosis of Herpes Simplex Encephalitis: Presentation of 5 Atypical Cases RENAN B. DOMINGUES', FRED D. LAKEMAN', CLAUD10 S. PANNUT13, MARIA CRISTINA D. FINK3 and ANA MARIA C. TSANACLIS' From the 'Experimental Neuropathology Laboratory, Department of Pathology, University of S6o Paulo, Brazil, the 'Department of Pediatrics, Microbiology, and Medicine, University of Alabama at Birmingham, USA, and the Virology Laboratory (LIM 52), Tropical Medicine Institute, S6o Paulo, Brazil

Four cases of herpes encephalitis (HSVE) are described. The diagnosis was established by polymerase chain reaction (PCR) assay of cerebrospinal fluid (CSF). These reports illustrate different situations in the clinical management of this disease. PCR was considered useful in confirming the HSVE diagnosis in 3 atypical cases, and in the differentiation between virologic failure and postinfectious encephalitis in a patient with recurrence of symptoms. A case with typical HSVE clinical findings is also reported where PCR was negative and a temporal lobe lymphoma was diagnosed at autopsy. This last case is representative of the utility of PCR in the management of other diseases mimicking HSVE.

R. B. Domingues, MD, R. Frei Caneca 617/apt. 94, Scio Paulo, SP 01307-001, Brazil

INTRODUCTION

Herpes simplex virus (HSV) is the most important etiology of focal encephalitis and can be detected in 45% of patients with focal encephalitis (1). Since the mortality of herpes encephalitis (HSVE) can be significantly reduced with acy- clovir treatment, an early and precise diagnosis is essential

Detection of HSV DNA by polymerase chain reaction in cerebrospinal fluid samples has been shown to be a very sensitive and specific method for herpes encephalitis (HSVE) diagnosis (3-6). In addition, some reports have suggested that PCR may reveal a wider clinical spectrum of HSVE than previously recognized (7).

This report describes the clinical presentation and PCR results of 5 cases of focal encephalitis and the importance of the application of the PCR to the diagnosis and manage- ment of these cases.

(2) .

METHODS AND PATIENTS PCR procedure Cerebrospinal fluid samples for PCR analysis were collected with disposable spinal tap needles. The specimens were aliquoted into 2 Cryo vial (NUNC, Roskilde, Denmark) sterile tubes in a separated room not used for viral isolation and stored at -20°C until used. One of the aliquots was sent to and processed in the Virology Laboratory, Tropical Medicine Institute of S9o Paulo, Brazil. The other CSF portion was shipped on dry ice to the Clinical Virology Laboratory, Department of Pediatrics, University of Alabama at Birmingham, USA. All the CSF manipulations before and during PCR procedure in both laboratories were performed in areas not used for viral isolation.

The sets of primers, the concentrations of the reagents in the PCR mixture, the cycles, the controls, and the detection of PCR products have been described previously (8). An identical protocol was used in both PCR laboratories. The HSV type was determined by DNA polymerase PCR products digestion with Hha I. HSV-I pol product has 1 cleavage site and HSV-2 has 2 cleavage sites with

this enzyme. The digestion products' sizes are 135 and 44 bp for HSV-1 and 76, 59, and 44 bp for HSV-2. Techniques to prevent contamination were adopted to avoid false-positive results (9).

Patient 1 A 59-year-old woman experienced fever and nausea. Three days later she presented bizarre behavior and 1 day later she had a decreased level of consciousness. On admission she was comatose with a Glasgow score of 6 and hemiplegic at the right side. CT scan showed a hypodense lesion in the left medial temporal lobe, with mass effect and contrast enhancement. CSF revealed 475 WBC/pl with 87% lymphocytes, protein 550 mg/l, glucose 900 mg/ 1. PCR for HSV DNA was positive and restriction enzyme analysis of PCR products indicated an HSV-1 infection. On the fourth day of illness, acyclovir was introduced (30 mg/kg/day); however, she had neurologic deterioration and 1 week after admission she died. The autopsy showed an extensive necrotic and hemorrhagic en- cephalitis involving both temporal lobes. HSV infection was con- firmed by brain tissue immunohistochemistry.

Patient 2 A 48-year-old man developed fever, headache and vomiting. One week later he was confused and at admission he had right hemi- paresis and a mixed aphasia with preserved consciousness. CT scan revealed an extensive lesion from the medial temporal left region to the left occipital lobe, with contrast enhancement. EEG showed periodic activity over the occipital left area. CSF showed 373 WBC/pl with 72% of lymphocytes, protein 380 mg/l and glu- cose 640mg/l. PCR results were positive for HSV-1 DNA. The patient was treated for 2 weeks with intravenous acyclovir (30 mg/ kg/day) and 6 months later he had regained normal language and motor functions.

Patient 3 A 92-year-old woman had fever and bizarre behavior. The next day she presented focal seizures, and remained comatose. On admission she was comatose, Glasgow score was 8 and she had right hemiplegia. The CT scan showed only cortical atrophy. CSF revealed 4 WBC/pI, protein 680 mg/l, glucose 740 mg/l. EEG showed periodic sharp waves over the left temporal and frontal regions. Phenytoin and acyclovir (30 mg/kg/day) were introduced

0 1997 Scandinavian University Press. ISSN 0036-5548

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Scand J Infect Dis 29 230 R. B. Domingues et al.

despite the fact that CSF PCR for HSV DNA was negative. There was a progressive clinical deterioration and 5 days after admission the patient died. The autopsy revealed primary lymphoma in the temporal lobe.

Patient 4 A 16-year-old boy complained of headache and fever. Two days later he became confused and he had a seizure. CSF revealed 160 WBC/pI, 97% lymphocytes, glucose 800 mg/l and protein 630 mg/l. CT scan and MRI revealed a lesion in the right temporal lobe, EEG showed slow waves over the temporal and posterior areas of the right hemisphere. The first CSF sample was strongly PCR positive for HSV-1. Acyclovir (30 mg/kg/day) and phenytoin were introduced. After 3 weeks of acyclovir treatment, his neuro- logic examination was normal and he returned to routine activities. Two weeks later he presented personality change and new seizures were observed. He was admitted with a mildly decreased level of consciousness. A new CSF sample revealed 16 WBCjpl, protein 660mg/l. HSV PCR results were negative on this second CSF sample. A second MRI showed more extensive T,-weighted hyper- dense lesions than the first MRI, involving the right temporal lobe and peri-ventricular white matter region. Although acyclovir (45 mg/kg/day) was re-introduced, this patient’s behavior became progressively more bizarre, including hypersexuality, hyperphagia and humor fluctuations. The progression of symptoms was inter- rupted with 1 mg/kg/day of Prednisone, but the patient remained with severe neurologic sequelae.

Patient 5

A 22-year-old woman complained of headache and fever. One day later she had a seizure. At admission she was somnolent with no focal neurological signs. There was no evidence of previous epilepsy. A contrast enhanced CT scan was normal. EEG revealed right temporal spikes. CSF analysis showed 117 WBCjpl, protein 282mg/l and glucose 600mg/l. CSF PCR was positive and the restriction enzyme products were compatible with HSV-2 infection. Acyclovir (30 mg/kg/day) and phenytoin were introduced. After 14 days of acyclovir therapy the patient was discharged. An MRI was performed at that time and was normal. There were no neurologi- cal symptoms during a 6-months follow-up.

DISCUSSION

All patients included in this series presented with a focal encephalitis. Different clinical presentations were observed for each patient. They illustrate different situations fre- quently found in clinical practice. CSF analysis by PCR for detection of HSV DNA was used to confirm the diagnosis of HSVE.

Soong et al. demonstrated that the most significant find- ings associated with HSVE are localization at EEG, CT, or technetium brain scan, age above 30 years and CSF WBC > 5 pl (10). Patient 1 presented all these findings. Although antiviral therapy was introduced, the disease progressed and the patient died. In this case, HSV DNA detection in CSF sample by PCR precluded further investi- gation, especially brain biopsy. Clinical presentation in case 2 was also suggestive of HSVE; however, there were some atypical features. A remarkable involvement of the left occipital lobe was seen on CT scan and spikes were regis- tered on EEG at left occipital area. There is a previous report of a patient with an occipital encephalitic focus

associated with HSV; however, this is a very unusual localization for the disease process (11). In case 3, the presence of fever, personality change, coma, and sharp waves at right temporal lobe at EEG examination raised a very strong suspicion of HSVE. However, CSF PCR was negative for HSV and the autopsy revealed central nervous system lymphoma. As previously described in cases of brain lymphoma, CT scan did not point to the diagnosis of a CNS tumor (12, 13). Also, Whitley et al. have previously reported that primary lymphoma in the CNS is one of the tumor diseases that can mimic HSVE (1). Other neurologi- cal diseases can also present similar clinical findings of HSVE, even when more recent neurodiagnostic methods are used (14).

Recurrence of clinical symptoms after an acute HSVE can occur in approximately 5-10% of patients (15). In such cases, it is necessary to discriminate between relapse of HSVE due to virologic failure, and postinfectious en- cephalitis (16-18). In the first situation, a second course of higher doses of acyclovir is necessary, and in the second immunosuppressive therapy could be beneficial. Patient 4 had an HSVE confirmed by PCR and after antiviral ther- apy the patient was discharged without sequelae. Recur- rence of clinical symptoms occurred 2 weeks after the patient was discharged. Although only brain biopsy can distinguish between viral reactivation and an auto-immune process, a negative PCR suggested the diagnosis of postin- fectious encephalitis.

Patient number 5 presented with a mild encephalitis. A previous report has demonstrated that HSVE cases with mild encephalopathy exist and similar cases may be unrec- ognized if a noninvasive method of diagnosis is not avail- able (7). Restriction enzyme analysis indicated an infection caused by HSV-2. In a study by Aurelius et al. (19), 93 consecutive cases of HSVE were analyzed. HSV-2 etiology was estabilished in 6 patients by antibody and PCR assays. One of the HSV-2 encephalitis cases had mild symptoms, similar to the patient described here. These descriptions confirm that HSV-2 etiology must be sought in mild forms of encephalitis.

In this series, 5 different situations of PCR utility were illustrated. (i) In typical cases like case 1, PCR can be considered useful in confirming the clinical suspicion, even when antiviral therapy cannot be shown to be beneficial. Viral resistance should be suspected and studied in such cases (20). (ii) Atypical cases of HSVE have been desribed (21, 22). In such cases, a positive PCR can establish an early diagnosis, avoiding further expensive and invasive investiga- tion. (iii) Other diseases can mimic HSVE. When a negative PCR is obtained, brain biopsy should be considered to determine the precise diagnosis. (iv) Case 4 illustrates the potential utility of PCR for differentiation between virologic failure and postinfectious encephalitis in clinical recurrence of symptoms. (v) Patient 5 confirms that HSV-2 etiology can be found among patients with mild encephalitis.

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Scand J Infect Dis 29

The routine analysis of CSF by PCR for the detection of HSV DNA should be adopted since empirical treatment is most susceptible to error. In addition, prospective studies with PCR are still necessary to establish the extension of the clinical spectrum of this disease, for instance, in mild and non-focal forms of encephalitis.

ACKNOWLEDGEMENTS

This work was supported by the following institutions and grants: FAPESP (FundaqBo de Apoio a Pesquisa, SBo Paulo, Brazil), project 94/1776-0; CNPq (Conselho Nacional de Desenvolvimento a Pesquisa), contract 201330/95-4, Ministry of Science and Tech- nology, Brazil; contracts NOl-AI-15113, N01-AI-62554 and N01- AI-12667 from the Antiviral Research Branch of the National Institute of Allergy and Infectious Diseases, a grant from the Division of Research Resources (RR-032) from the National Insti- tutes of Health and a grant from the state of Alabama.

We gratefully acknowledge collaboration with the Departments of Neurology (Dr Paulo E Marchiori and Prof Dr Milberto Scaff) and Infectious Diseases (Prof Dr Vicente Amato Neto) of the University of SBo Paulo.

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Submitted 4 November 1996; accepted 23 February 1997

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