Journal of the Neurological Sciences, 109 (1992) 61-63 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00

61

JNS 03732

Interferon-3, in cerebrospinal fluid without pleocytosis in scrub typhus Masayuki Ikeda a, Shoji Yoshida b and Hiroshi Tsukagoshi c a Department of Neurology, b Department of Medicine, Asafii General Hospital, Asah~ Chiba, Japan, c Department of Neurology, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan (Received 30 April, 1991) (Revised, received 22 October, 1991) (Accepted 30 October, 1991)

Key words: Interferon-y; Scrub typhus; Cerebrospinal fluid; Meningitis; lntrathecal synthesis Summary

We detected immunoreactiveinterferon-3, (IFN-3,) both in cerebrospinal fluid (CSF) and in serum of 5 patients with scrub typhus, one with meningitis and 4 other cases with neither CSF pleocytosisnor blood-CSF barrier dysfunction. Our data suggest in~rathecal synthesis of IFN-y without pleocytosiswhich implies occult cerebral involvementin scrub typhus.

Introduction

Materials and methods

Fever and headache are always associated with scrub typhus (Tsutsugamushi disease) (Berman and Kundin 1973), but conventional cerebrospinal fluid (CSF) examination is often negative despite neck stiffness (Sayen and Pond 1946). However, previous pathological studies have confirmed leptomeningeal involvement in all cases examined (Sayen and Pond 1946; Well and Haymaker 1946). Therefore, there may be a meningeal response in scrub typhus which is undetected by standard CSF examination. Importance of Interferon-3, (IFN-3,), a major effecter involved in macrophage activation to kill rickettsiae, has been studied only in mice (Palmer et al. 1984; Jerrells et al. 1988) or non-human primates (MacMillan et al. 1985) in rickettsial infections. In order to investigate immunological cerebral involvement in human rickettsial infection we assayed IFN-3, both in CSF and serum samples from 5 patients (one with clinical meningitis and 4 with normal CSF examination) with scrub typhus. In addition, we estimated blood-CSF barrier (BCB) permeability to evaluate intrathecal IFN-3, synthesis.

Subjects We studied paired serum and CSF samples from 5 patients (all men, between 41 and 66 years of age, mean age 54 yrs) with scrub typhus and 10 age-matched control subjects (7 men and 3 women, between 36 and 70 years of age, mean 57 yrs) with non-inflammatory neurological diseases (3 with Parkinson's disease, 2 with peripheral neuropathy, 3 with cervical spondylosis, 2 with muscle contraction headach). Since all the patients with scrub typhus had fever and headache, lumbar puncture, performed non-traumatically, was indispensable to rule out meningitis. Serum samples were collected from each subject at the same time of the lumbar puncture. After a 2-week treatment by minocycline 200 mg/day, convalescent sera were also collected from all 5 patients with scrub typhus. Convalescent CSF was available only from the case with meningitis (case 1). The samples were stored at -80°C until assayed.

Correspondence to: Dr. Masayuki lkeda, Department of Neurology, School of Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo, Tokyo 113, Japan.

IFN-7 assay Levels of human IFN-7 were determined by immunoradiometric assay (IRMA) using Sucrosep IFN-7 IRMA (Celltech Diagnostics Ltd., Slough, U.K.) that was able to detect levels higher than 1.0 U / m l of human iFN-3,. Collection and handling of specimens were performed and a standard curve was generated in accordance with the manufacturer's recommendation

62 and the procedural details published previously (Abbott et al. 1987). Measurement was performed simultaneously on all the samples. The results were reproducible on repeated testing.

TABLE 2

Case No.

Acute

Evaluation of BCB permeability

(serotype)"

IgG

IgM

IgG

lgM

Serum and albumin levels were measured by laser nephelometer and a CSF/serum albumin quotient (normal range; < 6.5 × 10-3; Reiber 1986) was calculated to evaluate BCB permeability.

1 (Gilliam) 2 (Karp) 3 (Gilliam) 4 (Gilliam) 5 (Kate)

40 < 10 < 10 40 160

10 < 10 < 10 20 160

160 320 40 320 640

20 80 80 160 640

Serodiagnosis of scrub typhus

a Serotype of the highest titer.

Specific IgM and IgG antibody titers against the 3 known serotypes of Rickettsia tsutsugamushi, namely Gilliam, Karp and Kate, were assayed by an indirect immunofluorescent (IF) method (Bozeman and Elisberg 1963). The diagnosis was confirmed by a 4-fold or greater increase in the serum antibody titer (IgG) between the acute and convalescent samples.

Results

Clinically, all the patients with scrub typhus had fever and headache. Headache was severe in the meningitis case (case 1) and moderate in other four cases. A typical eschar and lymphoadenopathy were present in all the cases. Only case 1 showed overt neck stiffness. After administration of minocycline 200 rag/ day, all the patients showed reduced fever and resolution of symptoms within 24 h. Table 1 demonstrates levels of IFN-y and albumin in sera and CSFs from the patients and control subjects. In the patient group, CSF cell count and protein content were abnormal only in case 1. IFN-3, was detected both in sera and CSFs from all the patients with scrub typhus. The IFN-3, level in the CSF was

CHANGES OF TITERS OF IMMUNOFLUORESCENT ANTIBODY AGAINST RICKETTSIA TSUTSUGAMUSHI 1N SERA Convalescent

even higher than that of the serum in case 2. Considering the CSF/serum albumin quotient (Reiber 1986), BCB was intact except in case 1. Serum IFN-Twas detected only in case 1 after a 2-week treatment when the symptoms had completely disappeared. In the control group, IFN-7 was not detected in any of the serum or CSF samples. Other CSF values, i.e. cell counts, protein and albumin levels and CSF/serum albumin ratio, were all normal. Table 2 shows serum antibody titers. All the patients with scrub typhus showed a 4-fold or greater increase in the IgG antibody titer. No antibody was detected in any of the CSF samples.

Discussion

Using murine (Palmer et al. 1984; Jerrells et al. 1988) or sub-human primate ( MacMillan et al. 1985) models, many reports have demonstrated that IFN-y plays an important role in rickettsial infection, but it is not yet clear whether the human response to the infection is similar or not. This report is the first

TABLE 1 IFN-y TITERS AND ALBUMIN LEVELS IN SERUM AND CSF Case

Serum

No.

IFN-7 a (IU/ml)

ALB b mg/dl

Cell/ mm 3

Ic 1f 2 3 4 5 CON g

6.3 1.0 1.7 35 2.1 9.0 n,d.

3110 3 760 3 640 3 680 3 490 3 920 3 9 4 3 + 105

176 16 1 3 l 4 2.1 +0.3

a

b d • f g

CSF Protein (mg/dl) 73 58 19 32 36 27 25.4+3.4

IFN-3, IU/ml)

ALB (mg/dl)

QIFN

3.3 1.7 2.8 3.3 !.8 3.6 n.d.

43.6 32.8 10.7 18.2 20.5 15.8 14.4 + 2.0

0.52 1.42 1.65 0.09 0.86 0.40

IFN-y -- interferon-gamma. ALB ~ albumin, c Q l ~ -- CSF/serum concentration quotient of IFN-y. QAt.n ----CSF/serum concentration quotient of albumin. Normal range is < 6.5 × 10 -3. Acute period. Convalescent period. CON = control. Values are expressed as mean + SEM. n.d. -- not detected.

¢

QALB d ( X 10 -3) 14 8.7 2.9 4.9 5.9 4.0 4.6 + 0.5

63 documentation of IFN-~/both in sera and CSFs of patients with scrub typhus, suggesting that IFN-7 may play some role also in the human immune response to

Rickettsia tsutsugamushi. Several studies have demonstrated that CSF IFN-y is associated with infectious CNS disease (Abbott et ai. 1985; Abbott et al. 1987; Frei et al. 1988; Griffin et al. 1990). The relatively low molecular weight (2500030000 dalton) of IFN-3, requires cautious interpretation of the data because of non-specific leakage of IFN-3, from serum into CSF through damaged BCB in the case of an inflammatory CNS disease. However, in the present study, the normal quotient of CSF/serum albumin in cases 2-5 indicates intact BCB. Furthermore, even if the serum level is much higher (128.8 IU/ml) than those of our cases, IFN-y is not always detected in the CSF (Panitch et al. 1987). Thus CSF IFN-3~ in the present study is not simple leakage through BCB but suggests intrathecal synthesis. IFN-7 in the CSF without pleocytosis may be an immune expression of occult cerebral involvement in scrub typhus. Every patient with scrub typhus has fever and headache (Berman and Kundin 1973), but conventional CSF examination is often negative as in the present study. Sayen et al. (1946) reported that only 10 out of 25 cases with overt neck stiffness showed CSF pleocytosis while previous pathological reports (Sayen et al. 1946; Well and Haymaker 1946) have demonstrated that leptomeningeal involvement was present in all cases examined. The earliest reaction consisted of a more or less marked edema of pia-arachnoid (Weil and Haymaker 1946). In rickettsial infection, the circulating microorganisms enter the capillary endothelial cells where they multiply. The vascular lesion may well involve leptomeninx and induce a local immune response. Direct invasion of the CNS by the rickettsiae could result in local production of IFN-7. Another explanation for IFN-3, in the CSF is some allergic reaction in the CNS associated with systemic infection. The failure to detect specific antibody in the CSF suggests the latter possibility but the significance of rickettsial antibody in the CSF remains to be established. Abbott et al. (1987) detected IFN-3, in the CSF without pleocytosis in 7 patients with viral infection. They suggested that generalised viral infection with meningeal irritation might lead to local IFN-3, production in the absence of an inflammatory meningeal

response. Interestingly, exogenous interferon administrated to normal volunteers causes fever, headache and sometimes neck stiffness (Scott et al. 1981). Further study is needed to elucidate the precise role of IFN-3~ in scrub typhus. Acknowledgement We are grateful to Mr. Michael Robinson for the critical reading of the manuscript.

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