Journal of Medical Virology 87:1235–1240 (2014)
Presence of Herpesvirus DNA in Cerebrospinal Fluid of Patients With Tick-Borne Encephalitis and Enteroviral Meningoencephalitis Kla´ra Labska´,1,2* Katerˇina Roubalova´,2 Dusˇan Pı´cha,1 and Vilma Maresˇova´1 1
1st Department of Infectious Diseases, 2nd Faculty of Medicine, Charles University in Prague and Bulovka University Hospital, Czech Republic 2 National Institute of Public Health, Centre for Epidemiology and Microbiology, Prague, Czech Republic
Reactivation of HHVs in the CNS due to inflammation has not been well described yet. The primary aim of this study was to investigate the frequency of HHV DNA detection in the cerebrospinal fluid (CSF) of immunocompetent patients with meningoencephalitis of other than HHV origin. The secondary aim of this study was to evaluate the impact of herpesvirus co-infection on the clinical course and patient outcome. Ninety-six patients with clinically and laboratory proven tick-borne encephalitis (TBE) and 77 patients with a confirmed diagnosis of enteroviral meningitis (EVM), along with a control group of 107 patients without evidence of inflammation in the CSF were retrospectively tested by nested PCR for the presence of DNA of the neurotropic herpesviruses HSV1, HSV2, VZV, and HHV6 in the CSF. The clinical course, laboratory tests, antiviral treatment, and neurological complications in a 6-month follow-up were compared between the groups positive or negative for HHV DNA in the CSF. HHV DNA was found in the CSF of 12 (6.9%) patients (6.3% and 7.8% in the TBE and EVM groups, respectively) and in 1 (0.9%) control patient. None of the patients had recent blisters or rash. The clinical course was comparably mild in all patients. No permanent neurological sequelae were observed. Only the CSF total protein level was significantly higher in HHV DNA-positive than in HHV-negative patients. J. Med. Virol. 87:1235–1240, 2015. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
herpesvirus; cerebrospinal fluid; tick-borne encephalitis; enteroviral meningoencephalitis
C 2015 WILEY PERIODICALS, INC.
INTRODUCTION Human herpesviridae (HHVs) are well-known pathogens with the ability to establish lifelong latency in humans. Primary infection mostly occurs during childhood. In adult population, the seropositivity reaches 60% (Human Cytomegalovirus, CMV) up to more than 90% (Epstein-Baar virus, EBV; Herpes Simplex virus 1, HSV1; Varicella-Zoster virus, VZV; and Human Herpesvirus 6, HHV6). Reactivations from latency or reinfections are frequent. All human herpesviruses are capable of invading the central nervous system (CNS) and persisting longitudinally in the brain [Chan et al., 1999; Theil et al., 2004]. Most of them may be associated with various acute, subacute, or chronic neurological diseases in both immunocompetent and immunocompromised patients [Cinque et al., 2003; Boivin 2004; De Bolle et al., 2005; Gilden et al., 2007; Dupuis et al., 2011; Becerra et al., 2013]. HSV1, HSV2, VZV, and HHV6 show the strongest neurotropism among HHVs and establish latency in the nervous tissue (HHV1, HHV2, and VZV in neurons of dorsal root ganglia, HHV6 in several cell types including glial cells, oligodendrocytes, and astrocytes [Liedtke et al., 1993; De Bolle et al., 2005; Persson et al., 2009]. Both primary infections and reactivations of these viruses can result in neurological sequelae. The mechanisms of HHV reactivation have not been well understood yet and may involve a large number of possible trigger Grant sponsor: Charles University Grant Agency; Grant number: 1166/2005-IV-GAUK. Correspondence to: Kla´ra Labska´, National Institute of Public Health, CEM, NRL for Herpetic Viruses, Sˇroba´rova 48, 100 42, Praha 10, Czech Republic. E-mail: [email protected] Accepted 29 January 2015 DOI 10.1002/jmv.24172 Published online 13 March 2015 in Wiley Online Library (wileyonlinelibrary.com).
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events. Inflammation is a well-known trigger of local HHVs reactivation [Kriesel et al., 1997; Reeves and Compton, 2011], but its contribution to the virus replication in the central nervous system is not clear. The detection of herpesviral DNA in the cerebrospinal fluid (CSF) by polymerase chain reaction (PCR) is considered as a diagnostic marker of active infection in the CNS [Aberle et al., 2005; Studahl et al., 2013; Venkatesan et al., 2013]. Since the introduction of PCR methods in CSF analysis, new HHV-associated neurological syndromes and atypical clinical presentations of previously known diseases have been identified [Gilden et al., 2007; Miller et al., 2013; Studahl et al., 2013]. Due to its sensitivity and promptness, the polymerase chain reaction has changed diagnostic and therapeutic approaches to herpesvirus infections and nowadays is included in many guidelines [Aberle et al., 2005; Kneen et al., 2012; Solomon et al., 2012; Steiner et al., 2010, 2012]. However, in some cases, the impact of HHV replication on neurological disease pathogenesis remains to be elucidated [Agut, 2011; Bhaskaran et al., 2013]. The primary aim of this study was to reveal possible co-infection with HSV1, HSV2, VZV, and HHV6 in the CNS of immunocompetent patients with aseptic (non-bacterial) meningitis or meningoencephalitis of other than HHV origin (tick-borne encephalitis, enteroviral meningitis/meningoencephalitis) based on the detection of viral DNA in the CSF by nested PCR. The secondary aim was to evaluate the impact of HHV co-infection, if any, on the clinical course of the disease and on the patient outcome. MATERIALS AND METHODS Patients and Samples The presence of herpesvirus DNA in the CSF was retrospectively examined in 77 patients with enteroviral meningitis (EVM) and 96 patients with tickborne encephalitis (TBE). The patients were treated at the Clinic of Infectious Diseases of the Na Bulovce Hospital, Prague. The diagnosis of aseptic meningitis/ meningoencephalitis was established based on the presence of typical clinical signs (meningeal irritation, headache, nausea, vomiting, tremor, ataxia, etc.) with CSF pleocytosis (more than 5 nucleated cells/mm3). The detection of enteroviral RNA in the CSF (Enteroviral consensus kit, Argene Biosoft, Verniolle, France) and evidence of virus- specific IgG and IgM antibodies (EIA anti-TBEV IgG and IgM, Euroimmun, Lu¨beck, Germany) in the serum and CSF were used for differential diagnosis of EVM and TBE, respectively. Demographic data are shown in Table I. Immunocompetence was defined in this study as the absence of autoimmune disease, malignancy, HIV positivity, or immunosuppressive treatment. The patients were followed up during their hospital stay and at months 1, 3, and 6 after discharge. On admission, the patients were examined for body J. Med. Virol. DOI 10.1002/jmv
TABLE I. Demographic Data and HHV DNA Detection in Patients and Controls Study group
TBE
EVM
No. of patients Age in years
96 77 Average 40.5 (41) 13.3 (12) (median) Range 2–76 2–48 Sex Male 63 43 Female 33 34 Number of HSV1 1 1 patients with HSV2 0 2 HHV DNA VZV 4 0 in CSF HHV6 0 3 VZV þ HHV6 1 0
Controls 107 24 (16) 4–76 60 47 0 1 0 0 0
temperature, meningeal irritation signs, and neurological sequelae. Basic laboratory tests were performed (blood elements count, C-reactive protein, bilirubin, AST, ALT, CSF cell count, glycorrhachia, lactate, and CSF total protein). A lumbar puncture was carried out in most patients on admission. During the hospital stay, the length of febrile periods, duration of neurological manifestations, presence of skin lesions (rash or blisters), headache duration, and other subjective complaints were monitored. EEG was performed in all patients while in hospital and repeated at 1-, 3- and 6-month check-ups in those with persisting neurological abnormalities. In-hospital drug therapy included antioedematous drugs (mannitol 100%, dexamethasone 16%). Only three patients were treated with antivirals: two of them shortly after admission to the hospital and the third one started antiviral therapy on day 9 of the hospital stay due to genital herpes eruption. All three patients were negative for HHV DNA in CSF samples collected on admission. The control group consisted of 107 patients without any signs of inflammation in the CSF who underwent lumbar puncture for diagnostic purposes (lyme borreliosis seropositivity or meningism during febrile period). No observational data were collected in this group. CSF samples from patients were collected consecutively and stored at 50˚C. Samples and medical records were obtained with institutional ethics committee approval (No. 187 of 13 January 2004) (issued by the Ethics Committee of the Bulovka University Hospital). DNA Isolation and PCR Analysis DNA was isolated from 200 ml of CSF using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the body fluid protocol supplied by the manufacturer. DNA isolates were stored at 20˚C and tested within 1 week after isolation. DNAs of HSV1 and HSV2 were detected in multiplex nested PCR using primers derived from the UL-42
Presence of HV DNA in CSF of TBE and EVM Patients
and US-4 genes, respectively [Cassinotti et al., 1996]. The first PCR run was performed in 40 cycles (95˚C, 50 sec; 55˚C, 30 sec; 72˚C, 50 sec) with 10 pmol of the outer primers, 0.4 mmol/L each of four dNTPs (Nucleotide Mix Plus, Roche Diagnostics, Basel, Switzerland), and 1.5 mmol/L MgCl2, 1.25 U of TaqDNA polymerase in PCR buffer (Taqara-Bio, Schiga, Japan). In the second reaction (30 cycles, 95˚C, 50 sec; 65˚C, 30 sec; 72˚C, 50 sec), 5 pmol of the inner primers and 0.2 mmol/L of dNTPs were used. To detect VZV DNA, a part of the gB II gene was amplified in nested PCR using the method and primers described by Liedtke et al. [1993]. HHV6 DNA was examined using the method designed by Kondo et al. [1993]. In nested PCR, a sequence from the major capsid protein gene, common for both HHV6A and B types, was amplified. In both the VZV and HHV6 PCR assays, 1.25 U of Taq-DNA polymerase with PCR buffer (Roche) were used and 0.5 U of uracyl-glycosylase (Roche) were added in the second PCR run to prevent cross-contamination with amplification products. Each PCR run contained a positive control (DNA isolate from the laboratory strain of the respective virus) and a negative control (a blind isolate). To avoid carry-over contamination, the samples were run in duplicates. All positive results were confirmed in another independent test. The assay sensitivities for HSV (2,000 and 3,000 genome equivalents (ge)/ml for HSV1 and 2, respectively, and 1,000 ge/ml for VZV) were established using QCMD proficiency panels. HHV6 assay sensitivity (100 ge/ml) was established using serial dilution of quantified positive control. PCR products were detected by agarose gel electrophoresis and ethidium bromide staining.
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was detected in the CSF of 12 patients (6.9%) suffering from aseptic EV meningitis/meningoencephalitis or TBEV encephalitis, while in the control group, only one patient (0.9%) tested positive for HHV DNA (P ¼ 0.006). The HHV DNA positivity rates were similar in the patients with TBE and EVM (6.3% and 7.8%, respectively, P ¼ 0.7). The presence of VZV DNA was found most often, that is, in five patients (2.9%), all of them suffering from TBE. HHV6 DNA was detected in four patients (2.3%), three of them with EVM and one with TBE. HSV1 and HSV2 DNAs were revealed in two patients (1.2%) each (one TBE patient carried HSV1 and three EVM patients HSV1 or 2). The co-presence of two herpesviruses (VZV and HHV6) was found in one patient with TBE. HSV2 DNA was detected in the only one control CSF sample from a patient without a known history of genital herpes. Only one patient had an eruption of genital herpes on day 9 of the hospital stay but CSF HHV DNA was not detected on admission. The rest of TBE or EVM patients involved in this study had no skin manifestation (blisters or rash) while in hospital. All patients had a mild form of the disease and no permanent sequelae were observed. Age, gender, clinical form of the disease, neurological complications, and EEG findings of the patients with HHV DNA in the CSF are depicted in Table II. The comparison of the clinical features and basic laboratory test results between HHV DNApositive and HHV DNA-negative TBE and EVM patients (Table III) did not indicate a significant impact of herpesvirus activation on the course of the underlying disease. Of the clinical characteristics studied, only the average CSF total protein level turned out to be significantly higher in HHV DNApositive patients.
Statistical Analysis The data collected for HHVs DNA-positive and negative patient groups were analyzed using the SPSS v.16 software. Binary and ordinal variables were analyzed using Fisher’s exact test and Pearson’s chi square test. The mean values of the categorical variables were evaluated using the Mann–Whitney U-test and t-test for two independent sample means. All results with a P value lower than 5% were considered statistically significant.
RESULTS The data collected for the TBE and EVM patients illustrate the well-known differences between the clinical courses of the two diseases. EVM patients were children and young adults (age median of 12 years) and 70% of them had pharyngitis. TBE patients were heterogeneous in age (median of 41 years) and expressed more meningoencephalitic forms of the disease, had longer febrile period, manifested leukocytosis in peripheral blood, and had more lymphocytes in the CSF. As shown in Table I, HHV DNA
DISCUSSION Previously, HHV reactivation was documented in inflammatory CNS diseases of other origin, i.e. multiple sclerosis [Chapenko et al., 2003; Sotelo et al., 2008] and Alzheimer disease [Mori et al., 2004]. Till now, little has been known about herpesvirus coinfection in neuroinfections caused by other infectious agents. This study provides evidence that such coinfection may occur in some patients with TBE or EVM, although it seems to be a rather rare event. The HHV DNA in the CSF might result either from latent virus genomes released from damaged cells or from active virus replication. As no signs of pronounced brain damage were found in the study patients, the latter alternative appears to be more probable. However, in the case of HHV6, the occurrence of chromosomally integrated virus cannot be ruled out. In the control CSF samples from the patients without markers of CNS inflammation, the presence of HHV DNA was significantly less frequent, thus indicating that an inflammatory process associated J. Med. Virol. DOI 10.1002/jmv
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TABLE II. Characteristics and Clinical Course of the Disease in Patients With HHV DNA in CSF Patient Age no. (years) Sex
HHV DNA in CSF
1 2
61 32
M M
VZV VZV
3
56
F
VZV
4
32
M
5
52
F
6
35
M
7
12
F
8 9 10 11 12
14 24 6 11 6
F F M M M
Underlying disease
Neurological sequelae
Tick-borne meningitis Tick-borne meningitis
Tick-borne meningoencephalitis VZV Tick-borne meningoencephalomyelitis VZV þ HHV6 Tick-borne meningoencephalitis HSV1 Tick-borne meningoencephalitis HSV1 Enteroviral meningoencephalitis HSV2 Enteroviral meningitis HSV2 Enteroviral meningitis HHV6 Enteroviral meningitis HHV6 Enteroviral meningitis HHV6 Enteroviral meningitis
with TBE or EV infection could trigger herpesvirus reactivation. The presence of herpesvirus DNA in the CSF due to asymptomatic herpesvirus reactivation/reinfection preceding TBEV or EV infection cannot be ruled out either, although the probability of such a DNA catching is low. Recent primary herpesvirus infection in the HHV DNA-positive patients is not probable, as none of them had clinical signs of acute herpesvirus infection including blisters, varicella, or rash at the time of TBE or EVM diagnosis. Moreover, the population seroprevalence data indicate that at their age, the vulnerability to the primary infection is very low [Roubalova´ and Seeman, 1998]. Conversely, in two other patients with a history of symptomatic herpes (one with HSV1 eruptions preceding the diagnosis and the other with HSV2 genital lesion noted on day 9 after diagnosis), HSV DNA was not detected in the CSF at the time of TBE diagnosis. Of the HHVs studied, VZV reactivated most often. This virus was more likely to be detected in older patients whose decreased immune control of VZV latency has been known to play a role in the reactivation of the virus. None of them had a history of herpes zoster. Similar results have been reported by others [Koskiniemi et al., 2001; Persson et al., 2009]. On the other hand, the prevalence of HHV6 DNA in children and that of HSV2 in young adults may reflect the tendency of these viruses to reactivate more frequently near to the time of primary infection [Wald 2004]. In children, a stimulatory effect of other herpesvirus infections, typical for this age category, may exert its influence, too. Despite the herpesvirus co-infection, all the patients with TBE or EVM had a mild form of the disease and no permanent sequelae. J. Med. Virol. DOI 10.1002/jmv
EEG finding
None Transient tremor and prolonged headache None
No abnormalities Mild diffuse abnormality
Transient paraparesis of the lower extremities Prolonged headache and abnormality of sleep None
Mild diffuse abnormality Mild diffuse abnormality
None
No abnormalities
Transient headache Transient tremor None None None
No abnormalities Mild diffuse abnormality Mild diffuse abnormality Mild diffuse abnormality Moderate diffuse abnormality
Mild diffuse abnormality
No abnormalities
Antibody response to the herpesviruses studied was not assessed due to its low predictive value in detecting herpesvirus reactivation. Neither was tested the virus-specific intrathecal antibody production, because this test is considered to be reliable from day 7 to 10 after the onset of the disease [Studahl et al., 2013], but in the study patients, a lumbar puncture was performed on day 4 (median). There was no need for a second lumbar puncture due to the quick recovery of all patients. Co-infections with a herpesvirus and another etiological agent of acute CNS disease were observed both in immunocompromised and immunocompetent patients [Park et al., 2002; Weinberg et al., 2005; Prete et al., 2009; Nahdi et al., 2012]. In some cases, the co-infection was associated with more pronounced signs of inflammation and a worse course of the disease. However, in the study group of patients, no significant differences were observed in the clinical course of the disease between those with HHV DNA in the CSF and the rest. The CSF total protein level was higher in the HHV-positive patients (P ¼ 0.013), but still in the range considered typical for aseptic meningitis. Our results indicate that in immunocompetent patients with EVM or TBE, herpesvirus reactivation is rather a bystander event, which has no significant impact on the course of the underlying CNS disease. Secondary herpesvirus reactivation due to inflammation in the CNS may complicate the interpretation of virus DNA findings in the CSF in relation to disease causality. For the diagnosis and treatment decisions, PCR results must be interpreted only in the context of the patient’s medical history, clinical symptoms, and results of other laboratory examinations. High virus
Presence of HV DNA in CSF of TBE and EVM Patients
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TABLE III. Comparison of Clinical Courses of TBE and EVN Between Patients Positive and Negative for HHV DNA in CSF TBE
a
Age (years) Length of hospital stay (days)a Body tem. > 37˚C (days)a Headache on admissionb Headache duration (days)a Vomiting on admissionb Peripheral blood WBC count (109/L) on admissiona Peripheral blood C-reactive protein (mg/L) on admissiona CSF lymphocyte count/mla CSF neutrophil count/mla CSF total protein (g/L)a Meningeal irritation on admissionb Signs of encephalitis on admissionb Paresis on admissionb EEG at 1 month after dischargec
EEG at 3 month after dischargec
EEG at 6 month after dischargec
Objec. neur. sequelae
Subjective complaints
1 3 6 1 3 6
month after discharged months after discharged months after discharged month after discharged months after discharged months after discharged
HHV DNA-positive (n ¼ 7)
HHV DNA-negative (n ¼ 89)
43 (32–61) 14 (9–18)
EVM
P
HHV DNA-positive (n ¼ 6)
HHV DNA-negative (n ¼ 71)
P
40 (6–76) 14 (5–32)
0.57 0.5
12 (6–24) 10 (8–13)
13 (2–48) 9 (6–19)
0.73 0.82
5.3 2.4 (SD) 7 (100%) 5 3.4 (SD) 2 (29%) 11.5 (8.4–15.5)
4.5 1.9 (SD) 83 (93%) 4.7 2.6 (SD) 35 (39%) 11.7 (4 - 24.6)
0.43 0.76 0.64 0.63 0.93
1.8 0.8 (SD) 6 (100%) 2.2 1.2 (SD) 4 (66%) 8.4 (5.4–11.5)
2.5 1.3 (SD) 70 (99%) 2.7 1.8 (SD) 47 (66%) 9.9 (4.3–16.9)
0.14 0.95 0.5 0.98 0.25
16.6 (2–90)
12.8 (6–81)
0.38
14.0 (4–22)
15.3 (0–74)
0.68
62.5 (29.7–99.3) 125.6 (2.7–4692.7) 28.7 (8.0–98.3) 43.7 (0–290.7) 1.07 (0.87–1.19) 0.87 (0.35–1.9) 5 (71%) 62 (70%)
0.71 73.3 (16.7–152) 100.4 (4–786) 0.89 67.1 (0.67–167.3) 42.9 (0–384) 0.01 0.6 (0.44–1.18) 0.48 (0.19–1.13) 0.93 6 (100%) 69 (97%)
0.77 0.36 0.14 0.9
4 (57%)
58 (65%)
0.72
1 (17%)
22 (31%)
0.56
1 (14%) 0: 1/1
4 (4%) 0: 4/8 1: 3/8 2: 1/8 0: 16/27 1: 11/27 2: 0/27 0: 21/29 1: 7/29 2: 1/29 40/77 (52%) 11/43 (26%) 6/35 (17%) 19/77 (25%) 8/43 (19%) 7/35 (20%)
0.67 1.0
0 None 1: 5 2: 1 0: 0/2 1: 2/2 2: 0/2 0: 1/2 1: 1/2 2: 0/2 1/6 (17%) 0/2 0/2 1/6 (17%) 0/2 0/2
0 0: 1
1.0 1.0
0: 7/30 1: 22/30 2: 1/30 0: 7/23 1: 15/23 2: 1/23 6/61 (10%) 2/42 (5%) 0/28 7/61 (11%) 4/42 (10%) 3/28 (11%)
0.53
0: 2/3 1: 1/3 2: 0/3 0: 1/2 1: 1/2 2: 0/2 2/4 (50%) 3/3 (100%) 0/3 1/4 (25%) 1/3 (33%) 2/3 (67%)
0.81
0.54
0.94 0.46 0.63 0.99 0.67 0.18
0.55
0.78 0.91 1.0 0.83 0.82 0.8
a
Mean (range). No. of patients (%). EEG rating scale 0–2 (0: normal, 1: moderate, 2: large), no. of patients/no. of patients examined, diffuse abnormalities only. d No. of positive patients/no. of patients examined (%), significant values of P < 0.05 in bold. b c
DNA loads in the CSF may indicate the patient is at high risk for clinically apparent herpesvirus reactivation [Aberle et al., 2005; Schloss et al., 2009]. The detection of HSV1 that is known to cause sporadic necrotising encephalitis should always be an indication for antiviral therapy. CONCLUSIONS HSV1, HSV2, VZV, or HHV6 DNA was detected in the CSF of 7% of the patients examined in the acute phase of enteroviral meningitis or tick-borne meningoencephalitis. The reason for HHV DNA presence was a self-limiting reactivation of the virus in CNS compartment during inflammation. In all patients,
the disease had a mild course without permanent sequelae. The presence of herpesviral DNA in the CSF had no significant influence on the disease outcome. Low frequency and negligible clinical impact of herpesvirus reactivation in TBE or EVM patients do not argue for routine herpesvirus testing in these diseases. For the interpretation of nucleic acid testing results in the CSF, bystander herpesvirus reactivation should be taken into account. REFERENCES Aberle SW, Aberle JH, Steininger C, Puchhammer-Sto¨ckl E. 2005. Quantitative real time PCR detection of Varicella-zoster virus DNA in cerebrospinal fluid in patients with neurological disease. Med Microbiol Immunol 194:7–12.
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Presence of Herpesvirus DNA in Cerebrospinal Fluid of Patients With Tick-Borne Encephalitis and Enteroviral Meningoencephalitis Kla´ra Labska´,1,2* Katerˇina Roubalova´,2 Dusˇan Pı´cha,1 and Vilma Maresˇova´1 1
1st Department of Infectious Diseases, 2nd Faculty of Medicine, Charles University in Prague and Bulovka University Hospital, Czech Republic 2 National Institute of Public Health, Centre for Epidemiology and Microbiology, Prague, Czech Republic
Reactivation of HHVs in the CNS due to inflammation has not been well described yet. The primary aim of this study was to investigate the frequency of HHV DNA detection in the cerebrospinal fluid (CSF) of immunocompetent patients with meningoencephalitis of other than HHV origin. The secondary aim of this study was to evaluate the impact of herpesvirus co-infection on the clinical course and patient outcome. Ninety-six patients with clinically and laboratory proven tick-borne encephalitis (TBE) and 77 patients with a confirmed diagnosis of enteroviral meningitis (EVM), along with a control group of 107 patients without evidence of inflammation in the CSF were retrospectively tested by nested PCR for the presence of DNA of the neurotropic herpesviruses HSV1, HSV2, VZV, and HHV6 in the CSF. The clinical course, laboratory tests, antiviral treatment, and neurological complications in a 6-month follow-up were compared between the groups positive or negative for HHV DNA in the CSF. HHV DNA was found in the CSF of 12 (6.9%) patients (6.3% and 7.8% in the TBE and EVM groups, respectively) and in 1 (0.9%) control patient. None of the patients had recent blisters or rash. The clinical course was comparably mild in all patients. No permanent neurological sequelae were observed. Only the CSF total protein level was significantly higher in HHV DNA-positive than in HHV-negative patients. J. Med. Virol. 87:1235–1240, 2015. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
herpesvirus; cerebrospinal fluid; tick-borne encephalitis; enteroviral meningoencephalitis
C 2015 WILEY PERIODICALS, INC.
INTRODUCTION Human herpesviridae (HHVs) are well-known pathogens with the ability to establish lifelong latency in humans. Primary infection mostly occurs during childhood. In adult population, the seropositivity reaches 60% (Human Cytomegalovirus, CMV) up to more than 90% (Epstein-Baar virus, EBV; Herpes Simplex virus 1, HSV1; Varicella-Zoster virus, VZV; and Human Herpesvirus 6, HHV6). Reactivations from latency or reinfections are frequent. All human herpesviruses are capable of invading the central nervous system (CNS) and persisting longitudinally in the brain [Chan et al., 1999; Theil et al., 2004]. Most of them may be associated with various acute, subacute, or chronic neurological diseases in both immunocompetent and immunocompromised patients [Cinque et al., 2003; Boivin 2004; De Bolle et al., 2005; Gilden et al., 2007; Dupuis et al., 2011; Becerra et al., 2013]. HSV1, HSV2, VZV, and HHV6 show the strongest neurotropism among HHVs and establish latency in the nervous tissue (HHV1, HHV2, and VZV in neurons of dorsal root ganglia, HHV6 in several cell types including glial cells, oligodendrocytes, and astrocytes [Liedtke et al., 1993; De Bolle et al., 2005; Persson et al., 2009]. Both primary infections and reactivations of these viruses can result in neurological sequelae. The mechanisms of HHV reactivation have not been well understood yet and may involve a large number of possible trigger Grant sponsor: Charles University Grant Agency; Grant number: 1166/2005-IV-GAUK. Correspondence to: Kla´ra Labska´, National Institute of Public Health, CEM, NRL for Herpetic Viruses, Sˇroba´rova 48, 100 42, Praha 10, Czech Republic. E-mail: [email protected] Accepted 29 January 2015 DOI 10.1002/jmv.24172 Published online 13 March 2015 in Wiley Online Library (wileyonlinelibrary.com).
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events. Inflammation is a well-known trigger of local HHVs reactivation [Kriesel et al., 1997; Reeves and Compton, 2011], but its contribution to the virus replication in the central nervous system is not clear. The detection of herpesviral DNA in the cerebrospinal fluid (CSF) by polymerase chain reaction (PCR) is considered as a diagnostic marker of active infection in the CNS [Aberle et al., 2005; Studahl et al., 2013; Venkatesan et al., 2013]. Since the introduction of PCR methods in CSF analysis, new HHV-associated neurological syndromes and atypical clinical presentations of previously known diseases have been identified [Gilden et al., 2007; Miller et al., 2013; Studahl et al., 2013]. Due to its sensitivity and promptness, the polymerase chain reaction has changed diagnostic and therapeutic approaches to herpesvirus infections and nowadays is included in many guidelines [Aberle et al., 2005; Kneen et al., 2012; Solomon et al., 2012; Steiner et al., 2010, 2012]. However, in some cases, the impact of HHV replication on neurological disease pathogenesis remains to be elucidated [Agut, 2011; Bhaskaran et al., 2013]. The primary aim of this study was to reveal possible co-infection with HSV1, HSV2, VZV, and HHV6 in the CNS of immunocompetent patients with aseptic (non-bacterial) meningitis or meningoencephalitis of other than HHV origin (tick-borne encephalitis, enteroviral meningitis/meningoencephalitis) based on the detection of viral DNA in the CSF by nested PCR. The secondary aim was to evaluate the impact of HHV co-infection, if any, on the clinical course of the disease and on the patient outcome. MATERIALS AND METHODS Patients and Samples The presence of herpesvirus DNA in the CSF was retrospectively examined in 77 patients with enteroviral meningitis (EVM) and 96 patients with tickborne encephalitis (TBE). The patients were treated at the Clinic of Infectious Diseases of the Na Bulovce Hospital, Prague. The diagnosis of aseptic meningitis/ meningoencephalitis was established based on the presence of typical clinical signs (meningeal irritation, headache, nausea, vomiting, tremor, ataxia, etc.) with CSF pleocytosis (more than 5 nucleated cells/mm3). The detection of enteroviral RNA in the CSF (Enteroviral consensus kit, Argene Biosoft, Verniolle, France) and evidence of virus- specific IgG and IgM antibodies (EIA anti-TBEV IgG and IgM, Euroimmun, Lu¨beck, Germany) in the serum and CSF were used for differential diagnosis of EVM and TBE, respectively. Demographic data are shown in Table I. Immunocompetence was defined in this study as the absence of autoimmune disease, malignancy, HIV positivity, or immunosuppressive treatment. The patients were followed up during their hospital stay and at months 1, 3, and 6 after discharge. On admission, the patients were examined for body J. Med. Virol. DOI 10.1002/jmv
TABLE I. Demographic Data and HHV DNA Detection in Patients and Controls Study group
TBE
EVM
No. of patients Age in years
96 77 Average 40.5 (41) 13.3 (12) (median) Range 2–76 2–48 Sex Male 63 43 Female 33 34 Number of HSV1 1 1 patients with HSV2 0 2 HHV DNA VZV 4 0 in CSF HHV6 0 3 VZV þ HHV6 1 0
Controls 107 24 (16) 4–76 60 47 0 1 0 0 0
temperature, meningeal irritation signs, and neurological sequelae. Basic laboratory tests were performed (blood elements count, C-reactive protein, bilirubin, AST, ALT, CSF cell count, glycorrhachia, lactate, and CSF total protein). A lumbar puncture was carried out in most patients on admission. During the hospital stay, the length of febrile periods, duration of neurological manifestations, presence of skin lesions (rash or blisters), headache duration, and other subjective complaints were monitored. EEG was performed in all patients while in hospital and repeated at 1-, 3- and 6-month check-ups in those with persisting neurological abnormalities. In-hospital drug therapy included antioedematous drugs (mannitol 100%, dexamethasone 16%). Only three patients were treated with antivirals: two of them shortly after admission to the hospital and the third one started antiviral therapy on day 9 of the hospital stay due to genital herpes eruption. All three patients were negative for HHV DNA in CSF samples collected on admission. The control group consisted of 107 patients without any signs of inflammation in the CSF who underwent lumbar puncture for diagnostic purposes (lyme borreliosis seropositivity or meningism during febrile period). No observational data were collected in this group. CSF samples from patients were collected consecutively and stored at 50˚C. Samples and medical records were obtained with institutional ethics committee approval (No. 187 of 13 January 2004) (issued by the Ethics Committee of the Bulovka University Hospital). DNA Isolation and PCR Analysis DNA was isolated from 200 ml of CSF using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the body fluid protocol supplied by the manufacturer. DNA isolates were stored at 20˚C and tested within 1 week after isolation. DNAs of HSV1 and HSV2 were detected in multiplex nested PCR using primers derived from the UL-42
Presence of HV DNA in CSF of TBE and EVM Patients
and US-4 genes, respectively [Cassinotti et al., 1996]. The first PCR run was performed in 40 cycles (95˚C, 50 sec; 55˚C, 30 sec; 72˚C, 50 sec) with 10 pmol of the outer primers, 0.4 mmol/L each of four dNTPs (Nucleotide Mix Plus, Roche Diagnostics, Basel, Switzerland), and 1.5 mmol/L MgCl2, 1.25 U of TaqDNA polymerase in PCR buffer (Taqara-Bio, Schiga, Japan). In the second reaction (30 cycles, 95˚C, 50 sec; 65˚C, 30 sec; 72˚C, 50 sec), 5 pmol of the inner primers and 0.2 mmol/L of dNTPs were used. To detect VZV DNA, a part of the gB II gene was amplified in nested PCR using the method and primers described by Liedtke et al. [1993]. HHV6 DNA was examined using the method designed by Kondo et al. [1993]. In nested PCR, a sequence from the major capsid protein gene, common for both HHV6A and B types, was amplified. In both the VZV and HHV6 PCR assays, 1.25 U of Taq-DNA polymerase with PCR buffer (Roche) were used and 0.5 U of uracyl-glycosylase (Roche) were added in the second PCR run to prevent cross-contamination with amplification products. Each PCR run contained a positive control (DNA isolate from the laboratory strain of the respective virus) and a negative control (a blind isolate). To avoid carry-over contamination, the samples were run in duplicates. All positive results were confirmed in another independent test. The assay sensitivities for HSV (2,000 and 3,000 genome equivalents (ge)/ml for HSV1 and 2, respectively, and 1,000 ge/ml for VZV) were established using QCMD proficiency panels. HHV6 assay sensitivity (100 ge/ml) was established using serial dilution of quantified positive control. PCR products were detected by agarose gel electrophoresis and ethidium bromide staining.
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was detected in the CSF of 12 patients (6.9%) suffering from aseptic EV meningitis/meningoencephalitis or TBEV encephalitis, while in the control group, only one patient (0.9%) tested positive for HHV DNA (P ¼ 0.006). The HHV DNA positivity rates were similar in the patients with TBE and EVM (6.3% and 7.8%, respectively, P ¼ 0.7). The presence of VZV DNA was found most often, that is, in five patients (2.9%), all of them suffering from TBE. HHV6 DNA was detected in four patients (2.3%), three of them with EVM and one with TBE. HSV1 and HSV2 DNAs were revealed in two patients (1.2%) each (one TBE patient carried HSV1 and three EVM patients HSV1 or 2). The co-presence of two herpesviruses (VZV and HHV6) was found in one patient with TBE. HSV2 DNA was detected in the only one control CSF sample from a patient without a known history of genital herpes. Only one patient had an eruption of genital herpes on day 9 of the hospital stay but CSF HHV DNA was not detected on admission. The rest of TBE or EVM patients involved in this study had no skin manifestation (blisters or rash) while in hospital. All patients had a mild form of the disease and no permanent sequelae were observed. Age, gender, clinical form of the disease, neurological complications, and EEG findings of the patients with HHV DNA in the CSF are depicted in Table II. The comparison of the clinical features and basic laboratory test results between HHV DNApositive and HHV DNA-negative TBE and EVM patients (Table III) did not indicate a significant impact of herpesvirus activation on the course of the underlying disease. Of the clinical characteristics studied, only the average CSF total protein level turned out to be significantly higher in HHV DNApositive patients.
Statistical Analysis The data collected for HHVs DNA-positive and negative patient groups were analyzed using the SPSS v.16 software. Binary and ordinal variables were analyzed using Fisher’s exact test and Pearson’s chi square test. The mean values of the categorical variables were evaluated using the Mann–Whitney U-test and t-test for two independent sample means. All results with a P value lower than 5% were considered statistically significant.
RESULTS The data collected for the TBE and EVM patients illustrate the well-known differences between the clinical courses of the two diseases. EVM patients were children and young adults (age median of 12 years) and 70% of them had pharyngitis. TBE patients were heterogeneous in age (median of 41 years) and expressed more meningoencephalitic forms of the disease, had longer febrile period, manifested leukocytosis in peripheral blood, and had more lymphocytes in the CSF. As shown in Table I, HHV DNA
DISCUSSION Previously, HHV reactivation was documented in inflammatory CNS diseases of other origin, i.e. multiple sclerosis [Chapenko et al., 2003; Sotelo et al., 2008] and Alzheimer disease [Mori et al., 2004]. Till now, little has been known about herpesvirus coinfection in neuroinfections caused by other infectious agents. This study provides evidence that such coinfection may occur in some patients with TBE or EVM, although it seems to be a rather rare event. The HHV DNA in the CSF might result either from latent virus genomes released from damaged cells or from active virus replication. As no signs of pronounced brain damage were found in the study patients, the latter alternative appears to be more probable. However, in the case of HHV6, the occurrence of chromosomally integrated virus cannot be ruled out. In the control CSF samples from the patients without markers of CNS inflammation, the presence of HHV DNA was significantly less frequent, thus indicating that an inflammatory process associated J. Med. Virol. DOI 10.1002/jmv
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TABLE II. Characteristics and Clinical Course of the Disease in Patients With HHV DNA in CSF Patient Age no. (years) Sex
HHV DNA in CSF
1 2
61 32
M M
VZV VZV
3
56
F
VZV
4
32
M
5
52
F
6
35
M
7
12
F
8 9 10 11 12
14 24 6 11 6
F F M M M
Underlying disease
Neurological sequelae
Tick-borne meningitis Tick-borne meningitis
Tick-borne meningoencephalitis VZV Tick-borne meningoencephalomyelitis VZV þ HHV6 Tick-borne meningoencephalitis HSV1 Tick-borne meningoencephalitis HSV1 Enteroviral meningoencephalitis HSV2 Enteroviral meningitis HSV2 Enteroviral meningitis HHV6 Enteroviral meningitis HHV6 Enteroviral meningitis HHV6 Enteroviral meningitis
with TBE or EV infection could trigger herpesvirus reactivation. The presence of herpesvirus DNA in the CSF due to asymptomatic herpesvirus reactivation/reinfection preceding TBEV or EV infection cannot be ruled out either, although the probability of such a DNA catching is low. Recent primary herpesvirus infection in the HHV DNA-positive patients is not probable, as none of them had clinical signs of acute herpesvirus infection including blisters, varicella, or rash at the time of TBE or EVM diagnosis. Moreover, the population seroprevalence data indicate that at their age, the vulnerability to the primary infection is very low [Roubalova´ and Seeman, 1998]. Conversely, in two other patients with a history of symptomatic herpes (one with HSV1 eruptions preceding the diagnosis and the other with HSV2 genital lesion noted on day 9 after diagnosis), HSV DNA was not detected in the CSF at the time of TBE diagnosis. Of the HHVs studied, VZV reactivated most often. This virus was more likely to be detected in older patients whose decreased immune control of VZV latency has been known to play a role in the reactivation of the virus. None of them had a history of herpes zoster. Similar results have been reported by others [Koskiniemi et al., 2001; Persson et al., 2009]. On the other hand, the prevalence of HHV6 DNA in children and that of HSV2 in young adults may reflect the tendency of these viruses to reactivate more frequently near to the time of primary infection [Wald 2004]. In children, a stimulatory effect of other herpesvirus infections, typical for this age category, may exert its influence, too. Despite the herpesvirus co-infection, all the patients with TBE or EVM had a mild form of the disease and no permanent sequelae. J. Med. Virol. DOI 10.1002/jmv
EEG finding
None Transient tremor and prolonged headache None
No abnormalities Mild diffuse abnormality
Transient paraparesis of the lower extremities Prolonged headache and abnormality of sleep None
Mild diffuse abnormality Mild diffuse abnormality
None
No abnormalities
Transient headache Transient tremor None None None
No abnormalities Mild diffuse abnormality Mild diffuse abnormality Mild diffuse abnormality Moderate diffuse abnormality
Mild diffuse abnormality
No abnormalities
Antibody response to the herpesviruses studied was not assessed due to its low predictive value in detecting herpesvirus reactivation. Neither was tested the virus-specific intrathecal antibody production, because this test is considered to be reliable from day 7 to 10 after the onset of the disease [Studahl et al., 2013], but in the study patients, a lumbar puncture was performed on day 4 (median). There was no need for a second lumbar puncture due to the quick recovery of all patients. Co-infections with a herpesvirus and another etiological agent of acute CNS disease were observed both in immunocompromised and immunocompetent patients [Park et al., 2002; Weinberg et al., 2005; Prete et al., 2009; Nahdi et al., 2012]. In some cases, the co-infection was associated with more pronounced signs of inflammation and a worse course of the disease. However, in the study group of patients, no significant differences were observed in the clinical course of the disease between those with HHV DNA in the CSF and the rest. The CSF total protein level was higher in the HHV-positive patients (P ¼ 0.013), but still in the range considered typical for aseptic meningitis. Our results indicate that in immunocompetent patients with EVM or TBE, herpesvirus reactivation is rather a bystander event, which has no significant impact on the course of the underlying CNS disease. Secondary herpesvirus reactivation due to inflammation in the CNS may complicate the interpretation of virus DNA findings in the CSF in relation to disease causality. For the diagnosis and treatment decisions, PCR results must be interpreted only in the context of the patient’s medical history, clinical symptoms, and results of other laboratory examinations. High virus
Presence of HV DNA in CSF of TBE and EVM Patients
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TABLE III. Comparison of Clinical Courses of TBE and EVN Between Patients Positive and Negative for HHV DNA in CSF TBE
a
Age (years) Length of hospital stay (days)a Body tem. > 37˚C (days)a Headache on admissionb Headache duration (days)a Vomiting on admissionb Peripheral blood WBC count (109/L) on admissiona Peripheral blood C-reactive protein (mg/L) on admissiona CSF lymphocyte count/mla CSF neutrophil count/mla CSF total protein (g/L)a Meningeal irritation on admissionb Signs of encephalitis on admissionb Paresis on admissionb EEG at 1 month after dischargec
EEG at 3 month after dischargec
EEG at 6 month after dischargec
Objec. neur. sequelae
Subjective complaints
1 3 6 1 3 6
month after discharged months after discharged months after discharged month after discharged months after discharged months after discharged
HHV DNA-positive (n ¼ 7)
HHV DNA-negative (n ¼ 89)
43 (32–61) 14 (9–18)
EVM
P
HHV DNA-positive (n ¼ 6)
HHV DNA-negative (n ¼ 71)
P
40 (6–76) 14 (5–32)
0.57 0.5
12 (6–24) 10 (8–13)
13 (2–48) 9 (6–19)
0.73 0.82
5.3 2.4 (SD) 7 (100%) 5 3.4 (SD) 2 (29%) 11.5 (8.4–15.5)
4.5 1.9 (SD) 83 (93%) 4.7 2.6 (SD) 35 (39%) 11.7 (4 - 24.6)
0.43 0.76 0.64 0.63 0.93
1.8 0.8 (SD) 6 (100%) 2.2 1.2 (SD) 4 (66%) 8.4 (5.4–11.5)
2.5 1.3 (SD) 70 (99%) 2.7 1.8 (SD) 47 (66%) 9.9 (4.3–16.9)
0.14 0.95 0.5 0.98 0.25
16.6 (2–90)
12.8 (6–81)
0.38
14.0 (4–22)
15.3 (0–74)
0.68
62.5 (29.7–99.3) 125.6 (2.7–4692.7) 28.7 (8.0–98.3) 43.7 (0–290.7) 1.07 (0.87–1.19) 0.87 (0.35–1.9) 5 (71%) 62 (70%)
0.71 73.3 (16.7–152) 100.4 (4–786) 0.89 67.1 (0.67–167.3) 42.9 (0–384) 0.01 0.6 (0.44–1.18) 0.48 (0.19–1.13) 0.93 6 (100%) 69 (97%)
0.77 0.36 0.14 0.9
4 (57%)
58 (65%)
0.72
1 (17%)
22 (31%)
0.56
1 (14%) 0: 1/1
4 (4%) 0: 4/8 1: 3/8 2: 1/8 0: 16/27 1: 11/27 2: 0/27 0: 21/29 1: 7/29 2: 1/29 40/77 (52%) 11/43 (26%) 6/35 (17%) 19/77 (25%) 8/43 (19%) 7/35 (20%)
0.67 1.0
0 None 1: 5 2: 1 0: 0/2 1: 2/2 2: 0/2 0: 1/2 1: 1/2 2: 0/2 1/6 (17%) 0/2 0/2 1/6 (17%) 0/2 0/2
0 0: 1
1.0 1.0
0: 7/30 1: 22/30 2: 1/30 0: 7/23 1: 15/23 2: 1/23 6/61 (10%) 2/42 (5%) 0/28 7/61 (11%) 4/42 (10%) 3/28 (11%)
0.53
0: 2/3 1: 1/3 2: 0/3 0: 1/2 1: 1/2 2: 0/2 2/4 (50%) 3/3 (100%) 0/3 1/4 (25%) 1/3 (33%) 2/3 (67%)
0.81
0.54
0.94 0.46 0.63 0.99 0.67 0.18
0.55
0.78 0.91 1.0 0.83 0.82 0.8
a
Mean (range). No. of patients (%). EEG rating scale 0–2 (0: normal, 1: moderate, 2: large), no. of patients/no. of patients examined, diffuse abnormalities only. d No. of positive patients/no. of patients examined (%), significant values of P < 0.05 in bold. b c
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