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Impact of Herpes simplex virus load and red blood cells in cerebrospinal fluid upon herpes

Neurologists must be aware of the numerous conditions that may mimic encephalitis. Ninety single CSF samples were tested for HSV-1 IgM antibodies by ELISA test supplied by Dia Medix Corporation, USA. Among the 224 patients, 135 (60.3%) patients were identified as having aseptic meningitis (n = 70, 51.9%), encephalitis (n = 41, 30.4%) or meningoencephalitis (n = 24, 17.8%) at discharge. Nonviral causes of AME are treatable. The laboratory diagnosis of chronic infection necessitates discussion between the clinician and laboratory technician to allow triaging of testing. The mortality rate was 32.6% six months after HSE diagnosis. PCR (Polymerase Chain Reaction) analysis for herpes virus DNA was done on CSF sample in all patients.

Longer delay in acyclovir initiation tended to be associated with higher mortality but did not reach statistical significance (p=0.08). In neonates, herpes encephalitis may be caused by either HSV-1 or HSV-2. HSV load in CSF was neither associated with mortality (p=1.00) nor with morbidity (p=0.90). ADEM, acute disseminated encephalomyelitis; AFB, acid-fast bacillus; Ag, antigen; CSF, cerebrospinal fluid; CT, computed tomography; CTFV, Colorado tick fever virus; CXR, chest x-ray; DFA, direct fluorescent antibody; EBV, Epstein-Barr virus; HHV, human herpesvirus; HSV, herpes simplex virus; LCMV, lymphocytic choriomeningitis virus; MNCs, mononuclear cells; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; PMNs, polymorphonuclear leukocytes; PPD, purified protein derivative; TB, tuberculosis; VDRL, Venereal Disease Research Laboratory; VZV, varicella-zoster virus; WNV, West Nile virus. These data do not support measurement of HSV load at admission in patients with HSE. The encephalopathy appears mainly in the late stages of the disease leading to the so-called AIDS-related dementia complex which is a sub-cortical dementia characterized mainly by a cognitivo-motor syndrome, with memory loss, bradypsychia and bradykinesia. Despite the introduction of acyclovir treatment, mortality and persistent neurologic impairment due to HSV encephalitis (HSE) remain high [3,4]: 15 to 20% of patients with HSE die and about 60% of surviving patients develop long-term neurological sequels [1,5].

Associated disorders. Electroencephalogram showed general background slowing bilaterally, typical of viral encephalitis. Previous histopathological descriptions of HSE also showed a relationship between a high number of viral particles upon brain biopsy and poor disease outcome [7]. However, since HSV load in the cerebrospinal fluid (CSF) is not routinely determined, data on the impact of high HSV load upon HSE outcome are scarce and contradictory [8-11]. We conducted a retrospective study in patients diagnosed with HSE in northern France from 1998 to 2005. Upon presentation, a careful evaluation is important to quickly identify and address all evolving and impending emergent neurologic and systemic issues. Data were collected retrospectively from patient medical records using a standardized questionnaire.

Morbidity was assessed using the Glasgow Outcome Scale (GOS) divided into 5 categories (1: good recovery without neurologic impairment; 2: mild disability; 3: moderate disability; 4: severe disability; 5: death) [12,13]. The following informations were collected: i) at treatment initiation in the hospital: age, sex, alcohol consumption, severity of underlying disease, body temperature and Glasgow coma scale (GCS [13]); ii) during patient stay in the hospital: CSF cell counts prior to acyclovir initiation and time period between hospital admission, lumbar puncture and acyclovir initiation. Severity of the underlying disease was determined using the Knaus scale (A: normal health status; B: moderate limitation (impossible to work or study); C: severe limitation (need to have a third person in daily life); D: bedridden patient) and the MacCabe score (0: no fatal disease; 1: death in the next five years; 2: death in the following year) [14,15]. HSV load was measured in CSF of patients with HSE using a frozen sample taken before acyclovir treatment was initiated. We considered these patients as having encephalitis according to the clinical, EEG, CT, MRI and laboratory findings. When frozen CSF was available (n=30), we extracted HSV DNA using the QIAamp® DNA Blood Qiagen kit according to the manufacturer’s instructions. As an example, cytotoxic T cells that specifically lyse HSV-infected targets in vitro predominate at focal sites of infection in murine models [9].

When no CSF was available to extract DNA (n=13), we quantified the viral load directly on DNA that had been extracted on the date of sampling and stored frozen at −20°C. Then, quantification of the viral load was performed using the artus® HSV-1/2 LC PCR kit manufactured by Qiagen. This PCR kit uses real time PCR for quantification and contains a second system of heterologous amplification to monitor for the possible presence of inhibitors. Finally, when both NAA/Cr-PCr and Cho/Cr-PCr ratios are simultaneously outside the normal range (fourth pattern), they ­define a « double encephalopathy » [6, 7]. First, we used descriptive statistics to evaluate patient characteristics according to CSF sample availability. Secondly, we assessed univariate associations between HSV load (or other variables collected) and HSE outcome defined as mortality at six months and morbidity at six months (low morbidity: GOS 1, 2 and 3 versus high morbidity: GOS 4 and 5). Non-parametric tests were used for all comparisons.

Fisher’s exact test and Wilcoxon’s test were used to compare qualitative and quantitative variables, respectively. A significance threshold of 0.05 was retained. Statistical analysis was performed using SAS software (SAS 9.1, SAS Institute Inc., Cary, NC, USA). If mass effect from significant cerebral edema is noted, hyperosmolar therapy with the use of mannitol or hypertonic saline may be necessary. The care practices included CSF sampling for diagnosis and management of these patients with suspected HSE. HSV qualitative PCR were performed routinely for this diagnosis. We performed quantitative PCR retrospectively on the residual frozen CSF.

No additional sampling was necessary. This study was approved by the “Comité de Protection des Personnes Nord Ouest IV”, the ethic committee of Lille Universitary Hospital. Prodromal phase of 4-10 days with nonspecific symptoms was common in HSV and were characterized by fever and constitutional symptoms which was observed in 80% of our patients. Those patients belonged to a previously published cohort [16]. Frozen CSF samples were available for 43 of them. All of these were from patients with HSV-1 encephalitis. Characteristics of these patients are presented in Table .

Mortality at 6 months was quite high (32.6%) and severe morbidity (GOS score 4 and 5) was frequent (44.7%). MR images display brain atrophy. Included and not included patients did not differ for other demographic, clinical or biological main characteristics (data not shown). Characteristics usually linked to poor prognosis in the literature were found in our cohort., i.e. age, alcohol consumption, time period between admission and acyclovir introduction (Table , [1,2,5,6,17,18]). We first studied factors associated with mortality six months after HSE onset (Table ). Median CSF HSV load was 2.1 log10 copies/μL in patients who survived vs.


1.7 log10 copies/μl in patients who had died at six months (Figure ). Foremost is HSV and the need to start IV acyclovir as early as possible. Older age was significantly associated with mortality at six months (71 years in deceased patients vs. 59 years in patients who survived, p=0.03). There was a trend toward an association between mortality at six months and longer time between admission and start of acyclovir treatment (2 days in deceased patients vs. 0 day in patients who survived, p=0.08). We next studied factors associated with morbidity six months after HSE onset (Table ).

M.R. CSF HSV load distribution by GOS class is shown in Figure . Median HSV load in CSF was 2.0 log10 copies/μL in patients with low morbidity (GOS 1, 2 and 3) versus 1.8 log10 copies/μL in patients with high morbidity (GOS 4 and 5). The difference was not significant (p=0.95). Factors significantly associated with severe morbidity were: older age (64 vs. 57 in patients with low morbidity, p=0.04), delayed lumbar puncture (1 day vs. Astrocytes are also infected by the JC virus.

0 days, p=0.006), a worse Knaus score (A: 18.8% vs. 81.3%; B+C+D: 66.7% vs. 33.3%, p=0.004) and the presence of red blood cells in the CSF sample (49 cells/μL vs. 0, p=0.05). A predominance of lymphocytes in the CSF was associated with lower morbidity (42% in patients with high morbidity vs. 80% in those with low morbidity, p=0.04). We assessed factors associated with HSE outcome in 43 patients with symptomatic HSV encephalitis who sought care in northern France between 1998 and 2005.

To our knowledge, this cohort is the largest ever used for evaluating the impact of HSV load in CSF upon HSV encephalitis outcome. Moreover, we used a technique of quantitative real time PCR, limiting the risks of false positive samples. We found no association between HSV load and HSE outcome, nor in mortality or morbidity six months after HSE onset. Our results are consistent with other studies concerning factors usually associated with poor outcome in HSV encephalitis: older age, delayed initiation of acyclovir treatment and a high Knaus score [1,2,5,6,17,18]. Regarding the absence of association between CSF HSV load and HSE outcome, our results are in contrast to results reported by Dominges et al.[8]. However, in that study, HSV load was studied as a dichotomous variable with a cut-off of 100 copies/μL separating high and low viral load levels. In our study, we analyzed the relationship between viral load and outcome, introducing viral load as a continuous variable.

Our results are in agreement with Wildeman et al.[10], Ruzek et al.[11] and Hjalmarsson A et al. [19]. Kamei S et al., who evaluated serial changes of intrathecal viral loads by chemiluminescence assay and nested PCR, did not find any evident association between viral load and outcome either [20]. Our results are also concordant with the study of Schloss et al., comparing nested PCR to quantitative real-time PCR [21]. The metabolic anomalies described in literature are in agreement with our results. In our cohort, all patients were treated by acyclovir and all samples were collected before introduction of antiviral treatment. The absence of an association between HSE outcome and CSF HSV load could suggest that the pathogenicity of HSE may not be directly linked to extent of viral replication, but rather depends on the host response.

This hypothesis is consistent with the fact that IgG levels were described to be significantly higher in patients with good prognostic than in patients with poor outcome [19]. Surprisingly, in our study, we found a significant association between a high red cell count in CSF and severe morbidity. To our knowledge, this association had not been described previously. It can be hypothesized that a higher red cell count in CSF could reflect more severe brain cell necrosis, which may be due to an intense and inappropriate immunological response of the host to viral aggression. Some experimental data suggested that an intense and inappropriate inflammatory response could be harmful, and might explain the higher risk of morbidity [22,23]. However, this hypothesis remains to be further elucidated. Our study had several limitations.

First, although our sample size was greater than in other studies [8,10,11], the number of patients with available CSF samples was still small. This limitation is due to the low prevalence of HSE. Thus, due to the lack of statistical power, we may not have identified a potentially existing association between HSV load and HSE outcome. However, we were able to identify factors that are usually reported to be associated with poor HSV outcome in the literature [1,2,5,6]. Second, the risk of selection bias occurrence in our study was low. Although not all patients with HSE were enrolled, those with and without available CSF samples did not statistically differ in their main characteristics. We observed a higher mortality rate in patients with available CSF samples than in those without.

The reason for this difference in mortality is unclear. However, this did not bias our findings regarding the impact of HSV load on HSV outcome. Our own experience is in agreement with these findings. This is, however, unfeasible and unrealistic. Finally, it can be speculated that retrospective determination of HSV load in frozen samples is not accurate and may lead to the absence of an association between viral load and HSE outcome. However, Revello et al. showed the absence of genetic matter loss in samples frozen at −20°C [9].

Jerome et al., measured HSV loads in the same samples directly after sampling and after storage at 4°C for 16 months. HSV loads in freshly analyzed samples did not differ from those that had been stored at 4°C [24]. It is to note that in this last study, three positive specimens were negative upon retesting after storage. All these samples contained very low levels of virus even in the original quantitation and the authors estimated that the negative results may represent the inherent inaccuracy of quantitation of viral DNA in samples with low levels of virus rather than a true loss of viral DNA. In our study, we found 5 negative samples by quantitative PCR, although they were positive with qualitative PCR. We think that we encountered the same phenomenon as Jerome et al.: accurate quantitation of viral DNA is not possible in samples with low levels of virus. In addition, we sometimes had to work with low quantities of CSF due to limited availability of samples.

This occurred more frequently in patients whose samples gave negative results by quantitative PCR. In one of these 5 patients, enough CSF was available to re-perform a real time qualitative PCR, which was positive at the 39th cycle, corresponding to a low viral load. Thus, we can consider that these false negative results by quantitative PCR do not bias our results, because they effectively correspond to low HSV loads.