Dengue encephalitis in French Guiana

0 INSTITUT Paris 1998

Res. Viral.

PASTEUR~ELSEVIER

1998,

149, 235-238

CASE REPORT

Dengue encephalitis in French Guiana D. Hommel (l), A. Talarmin c2) (*), V. Deubel (3), J.M. Reynes (2), M .T . Drouet c3), J.L. Sarthou c2) and A. Hulin (l) (‘I Intensive Care Unit. General Hospital, Cayenne (French Guiana), f2JCentre National de RkjZrence pour la Surveillance des Arboviroses, Institut Pasteur de la Guyane, Cayenne (French Guiana), and (3)Arbovirus and Haemorrhagic Fever Viruses Unit, Institut Pasteur, Paris

SUMMARY

Thousands of cases of dengue fever (DF) and several cases of dengue haemorrhagic fever were recorded in French Guiana during the recent outbreak of dengue-2 virus (1991-1992) and in subsequent years. One case with clinical signs typical of classical DF with neurological complications is reported in this study. The neurological features (encephalitis) appeared during the acute phase, 2 days after the onset of fever. Dengue2 virus was detected in both the cerebrospinal fluid and blood sample. This case was fatal. This first reported case of classical DF with encephalitis in French Guiana is a new demonstration of the potential neurovirulence of dengue viruses. Key-words: Dengue, Encephalitis; French Guiana, Case report.

Dengue fever (DF) is regarded as the most important tropical arboviral disease in humans because of the degree of morbidity and mortality involved (Gubler, 1988). DF is also a major public health problem in French Guiana, an overseas French department (administrative unit) located between Brazil and Surinam, in the Amazonian forest. Indeed, an epidemic of dengue haemorrhagic fever (DHF) caused by dengue-2 was responsible for 40 DHF cases and 6 deaths in 1991 and 1992 (Reynes et al., 1994). This epidemic, almost entirely limited to the littoral area of French Guiana where 90% of the 136,000 inhabitants live, was followed by sporadic circulation of dengue viruses from 1993 to 1996.

Submitted

May

4, 1998,

accepted

(*) Corresponding author: A. Talarmin, Arboviroses pour la RBgion Antilles-Guyane,

July

Classical DF is characterized by sudden onset of fever, headache and retro-ocular pain, myalgia, arthralgia, nausea and a rash. The acute phase generally lasts for four to seven days, but long-term recovery, generally associated with asthenia, is common. Neurological complications are frequent with DHF (Sumarmo et al., 1983 ; George et al., 1984), but are rare in classical DF, although some cases have been reported in Australia (Row et al., 1996) and South-East Asia (Lum et al., 1996). We report in this study the first case of encephalitis associated with classical DF in French Guiana. This case was admitted to the intensive care unit (ICU) of Cayenne Hospital, French Guiana,

1, 1998.

Centre National de Refhrence pour la Surveillance Institut Pasteur de la Guyane, BP 6010, 97306

de la Dengue, de la Fikvre jaune Cayenne cedex, French Guiana.

et autres

D. HOMMEL

236

with neurological symptoms. Two techniques were used to detect antibodies to dengue viruses. Haemagglutination inhibition titres were determined using the method of Clarke and Casals adapted to microtechniques (Clarke and Casals, 1958), and dengue antibody responses were interpreted according to the WHO criteria. We also used the IgM capture enzyme immunoassay with a tetravalent dengue antigen in a procedure modified from that previously published (Chungue et al., 1989). For virus isolation, Aedes pseudoscutellaris (AP61) cell lines were incubated for 1 h with a 1: 10 dilution of acute phase serum or cerebrospinal fluid (CSF), rinsed and incubated for 7 days at 28°C in Leibowitz medium containing 3 % foetal calf serum. Dengue viruses were then identitied by indirect immunofluorescence assay using anti-dengue type-specific monoclonal antibodies (Henchal et al., 1983). For detection of dengue viruses by reverse transcription polymerase chain reaction (RTPCR), 20 ~1 of serum or CSF were mixed with lysis buffer (4 M guanidium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5 % sarkosyl, 100 mM P-mercaptoethanol) and phenol-chloroform mixture, as described previously (Chomczynski and Sacchi, 1987). RNA in the aqueous phase was precipitated with an equal volume of isopropanol. The first run of RT/PCR and subsequent semi-nested PCR were performed following the procedure of Lanciotti et al. (1992). Aedes aegypti male mosquitoes were inoculated intrathoracically with a 1: 10 dilution of serum or CSF and maintained for 2 weeks. Mosquitoes were ground in Hanks medium and supernatants were used to inoculate AP61 cells (Rosen and Gubler, 1974).

ET AL. nine, although blood smear for Plasmodium falciparum tested negative. Two days after onset of fever, he suffered a sudden cardio-respiratory arrest which was reversed. He was given tracheal intubation and ventilation support for cerebral protection and was transferred by air to Cayenne on the third day of his illness. On admission to the ICU, he presented with an encephalitis ; neurological tests showed that the patient was in a deep coma and had no cornea1 reflexes. The patient was given symptomatic neurological critical care treatment. Laboratory results on admission showed haematological disorders, haemoglobin 10.6 g/d1 ; haematocrit 34 % ; platelet count 85,000/~1 and biochemical disorders : sodium 120 mmol/l, calcium 1.8 mmol/l, urea 12.3 mmol/l, creatinine 23 1 mmol/l and aspartate transaminase 120 units/l. The results of other haematological, biochemical and haemostasis tests were normal and peripheral blood smears tested negative for malarial parasites. A lumbar puncture was performed. Less than 1 red blood cell/ml and less than 1 white blood cell/ml were detected. All bacterial cultures and serological tests were negative, except that dengue-2 was detected in both CSF and blood samples by RT-PCR. Virus isolation from CSF but not from serum was obtained only after virus amplification in A. aegypti male mosquitoes (table I). Samples tested negative by RT-PCR after the first run and positive after semi-nested PCR using the dengue-2specific internal primer (fig. 1). A computed tomography scan showed generalized cerebral oedema without focal lesions. Despite treatment, the neurological condition of the patient continued to deteriorate and he died 48 h after admission. Serological tests for dengue virus remained negative because of this rapid fatal outcome.

A 6-year-old boy was admitted to a local health centre in May 1995 with an altered level of consciousness and fever. He was initially treated symptomatically and with antibiotics and qui-

As the presence of virus in the central nervous system had not been demonstrated previously in reports of neurological manifestations in dengue infections, they were thought to be due to an encephalopathy secondary to DHF/DSS rather

CSF DF DHF

DSS ICU RT-PCR

= = =

cerebrospinal fluid. dengue fever. dengue haemorrhagic

fever.

= = =

dengue shock syndrome. intensive care unit. reverse transcription/polymerase

chain reaction.

DENGUE Table I. Detection

of dengue-2 virus and CSF of the patient.

Day after onset Mosquito cell culture (AP61) RTkemi-nested PCR Mosauito inoculation(*): AP61 cell culture

ENCEPHALITIS

CSF D6

-

-

+ -

+ +

237

GUIANA

(1996) detected dengue-2 and dengue-3 viruses in the CSF and blood of five patients with encephalitis, suggesting that these viruses can be neurovirulent.

in the serum

Serum D3

IN FRENCH

Our study shows that dengue-2 virus, detected in the CSF of a 6-year-old boy, was responsible for encephalitis. Very few red blood cells were present in the CSF, and the virus load was higher in the CSF, as in the blood (table I). Therefore, the presence of the virus in the CSF was probably not due to a traumatic lumbar puncture. A lack of white blood cells in the CSF, as reported for this patient, has previously been reported for two other patients (Lum al., 1996).

(*) Aedes aegJ@ male mosquitoes were inoculated intrathoracically with biological sample diluted 1: 10 and maintained at 30°C for two weeks before being ground in culture medium and inoculated into AP61 cell culture (Rosen and Gubler, 1974). At day 7 after infection, cells were tested by IFA using anti-dengue serotype-specific monoclonal antibodies (Henchal al., 1983).

et

et

In our patient, encephalopathy began before the third day of the illness ; therefore, the crossing of the blood-brain barrier was not due to increased vascular permeability or plasma leakage which generally occurs later in illness. The presence of the virus in the CSF so early in the disease is probably the best argument in favour of a true encephalitis by direct invasion of the brain by dengue viruses.

than to encephalitis. Neurological manifestations are common with DHF/DSS and are probably caused by prolonged shock, metabolic acidosis, electrolyte disturbance, liver failure, and disseminated intravascular coagulation leading to cerebral hypoxia and ischaemia (Nimmanitya et al., 1987 ; Hendarto and Hadenigoro, 1992). However, six cases of dengue encephalitis have recently been reported in southeast Asia (Lum et al., 1996).

Although the case of encephalitis reported in this study and others showed that dengue viruses can affect the brain, the neurotropism of dengue viruses has yet to be demonstrated. Indeed, cases of DF associated with neurological symptoms are

Attempts at isolating the virus from the brain and CSF were unsuccessful until Lum et al.

MW Dl

Serum

CSF

02 D3 D4 Dl

D2 D3 D4

119bD

Fig. 1. Agarose gel electrophoresis of the DNA products from RT-semi-nested PCR on RNA samples from dengue virus, directly extracted from serum and cerebrospinal fluid (CSF).

238

D. HOMMEL

’

rare, although millions of DF cases are reported worldwide. Encephalitis associated with DF may only be anecdotal and is only observed because of the major increase in the incidence of dengue over the past 40 years (Gubler, 1988). However, recent reports of dengue encephalitis may also be due to the development of sensitive methods for detecting dengue viruses, such as mosquito cell cultures, inoculation of mosquitoes and molecular biology. Our results confirm that a diagnosis of dengue fever should be considered in cases of encephalitis in tropical countries.

Acknowledgements This study was supported by a grant from the ProgrammeHospitalierde RechercheClinique.

Une endphalite due 1 la dengue en Guyane franqaise Durant l’Cpid&mie de dengue de 1991- 1992 due au virus dengue-2, et les antrees suivantes, des milliers de cas de dengue classique et de nombreux cas de dengue hemorragique ont CtC notifies en Guyane. Nous rapportons ici un cas de dengue classique avec complications neurologiques. Les signes endphalitiques sont apparus durant la phase prCcoce, deux jours aprbs le debut de maladie et l’issue fut fatale. Le virus dengue-2 a et6 detect6 dans le sang et le liquide cephalorachidien. Ce premier cas d’endphalite like a un virus de la dengue en Guyane franGaise est une nouvelle indication du pouvoir neuropathogkne de ces virus.

ET AL. References Chomczynski,P.& Sacchi,N. (1987),Single-step methodof RNA isolationby acid guanidiumthiocyanate-phenolchloroformextraction.Anal. Biochem., 162,156-159. Chungue,E., Boutin, J.P. & Roux, J. (1989) Inter&t du tirage des IgM par technique immunoenzymatique pour le serodiagnosticde la dengue en Polynesie franGaise.Rex Viral., 140,229-240. Clarke,D.H. & Casals,J. (1958),Techniquesfor hemagglutination and hemagglutination-inhibition with arthropod-borneviruses.Am. J. Trop. Med. Hyg., 7, 561-573. George,R., Gan, S.C. & Tuen, KS. (1984),Changingpattern in the clinical presentationof denguehaemorrhagic fever in Malaysia during the period 1962 to 1982.J. Malay. Sot. Health, 4, 57-64. Gubler, D.J. (1988), Dengue. in “The arboviruses: epidemiology and ecology” (J.P. Monath) (pp. 223260). CRC Press,Boca Raton,USA. Henchal, E.A., McCown, J.M., Seguin, M.C., Gentry, M.K. & Brandt, W.E. (1983) Rapid identification of denguevirus isolatesby usingmonoclonalantibodies in an indirect immunofluorescenceassay.Am. J. Trop. Med. Hyg., 32, 164-169. Hendarto, S.K. & Hadenigoro, S.R. (1992), Dengue encephalopathy.Acta Paediatr. Jpn., 34, 350-357. Lanciotti, R.S., Calisher,C.H., Gubler, D.J., Chang,G.J. & Vomdam,A.V. (1992),Rapiddetectionandtyping of dengue viruses from clinical samplesby using reverse transcriptase-polymerase chain reaction. J. Clin. Microbial., 30, 545-551. Lum, L.C.S., Lam, S.K., Choy, Y.S., George,R. & Harun, F. (1996), Dengueencephalitis: a true entity? Am. J. Trop. Med. Hyg., 54, 256-259.

Nimmanitya, S., Thisyakorn, U. & Hemsrichart, V. (1987),Denguehaemorragicfever with unusualmanifestations. Southeast Asian J. Trap. Med. Public Health, 18, 398-406.

Reynes, J.M., Laurent, A., Deubel, V., Telliam, E. & Moreau, J.P. (1994), The first epidemic of dengue hemorrhagicfever in French Guiana. Am. J. Trop. Med. Hyg., 51, 545-553. Rosen,L. & Gubler, D. (1974), The useof mosquitoesto detect and propagatedengueviruses. Am. J. Trop. Med. Hyg., 23, 1153-1160. Row, D., Weinstein,P. & Murray-Smith, S. (1996) Dengue fever with encephalopathyin Australia. Am. J. Trop. Med. Hyg., 54, 253-255.

Mets-cl&s : Dengue, EncCphalite franGaise, Rapport d’un cas.

; Guyane

Sumarmo,H.W., Jahja,E., Gubler, D.J., Subaryono,W. & Sorensen,K. (1983) Clinical observationson virologically confirmed fatal dengue infections in Jakarta,Indonesia.Bull. W.H.O., 61, 693-701.