Human Rabies in Israel.

To the Editor: Rabies, a major zoonotic disease in the Middle East, has two main epidemiologic forms: urban and sylvatic. The last case of human rabies in Israel was in the Golan Heights in 1971 (1). Twenty-five years later, in 1996, rabies was reported in a 20-year-old soldier, and then two cases were documented in 1997.

The first case-patient, a soldier in the Golan Heights, was bitten on the lip by an unidentified animal while sleeping. The wound was cleansed and sutured; clinical signs started 39 days later with high fever and headache. The patient was admitted to an emergency room with hallucinations, difficulty in swallowing, and generalized weakness, and rabies was considered in the differential diagnosis; 3 days later the patient became comatose. Samples of saliva, serum, and cerebrospinal fluid; skin biopsy tissue; and corneal impressions were sent to the Pasteur Institute, Paris, France. Eight days after clinical signs developed, rabies was diagnosed by the Kimron Institute by heminested reverse transcription-polymerase chain reaction (hnRT-PCR) on the patient's saliva (2). The RT step was performed with the specific primer 113 (5'-GTAGGATGATATATGGG-'3 at 1013-1030), followed by PCR with the 509 (5'-GAGAAAGA ACTTCAAGA-'3 at 1156-1173) and 304 (5'-GAGT CACTCGAATATGTC-'3 at 1513-1533) primers. The hn-PCR was performed with the 509 and 105 (5'-TTCTTATGAGTCACTCGAATA TGTCTTGTTTAG-'3 at 1393-1426) primers (3). The PCR results were confirmed by the Pasteur Institute 3 days later, and the patient died 35 days after clinical symptoms appeared.

The second case-patient was a 7-year-old girl admitted to the hospital unconscious. Two months before admission, she had been scratched while sleeping by an unidentified animal. On the second hospital day, generalized convulsions and gasping occurred. During the following days, brain stem function progressively deteriorated. Rabies was diagnosed by hnRT-PCR on the saliva sample, and the diagnosis was confirmed by the Centers for Disease Control and Prevention (CDC). The patient died despite supportive care.

The third case-patient, a 58-year-old man with fever, headache, and sore throat, was diagnosed as having pharyngitis and received an oral antibiotic. The patient had been bitten 3 months earlier while sleeping. On admission, the lumbar puncture, computerized tomography scan, and electroencephalogram were normal. On the third hospital day, he had respiratory arrest; during orotracheal intubation, acute laryngospasm with copious amounts of salivation occurred. Rabies was suspected, and viral RNA in the saliva was detected by hnRT-PCR. One day later the patient died.

We injected antemortem saliva and postmortem brain tissue from these patients into suckling mice intracerebrally. Virus was isolated from saliva samples of case-patients 1 and 3 but not from the sample of case-patient 2. Rabies virus antigen in the brain tissue was confirmed by direct immunofluorescence assay, and viral RNA was detected by RT-PC, R.

For genetic analysis, we used brain samples from the three case-patients and from animals that died of rabies near the location of the case-patients to amplify and sequence a 328-bp (264 bp from the 3' of the N gene and 64 bp of the 3' NS-N region) fragment. On the basis of homologic results of nucleotide sequences in the three case-patients and in virus isolates from animals in the same regions, we concluded that a reservoir for rabies in foxes is responsible for infection of all three humans.

The three human isolates were tested with a panel of 19 anti-N protein monoclonal antibodies (CDC, Atlanta, GA, USA) and compared with those of rabies isolates from the geographic vicinity of the human cases. Isolates from case-patients 1 and 3 belonged to variant 1 (MAb C18 negative) and were similar to virus isolates from 10 foxes, one jackal, and four cattle in the same regions. Isolates from case-patient 2 belonged to antigenic variant 2 (MAbs C2, C7, C 12, C13, C18 negative) and were similar to isolates from four foxes, one dog, and one cow in the vicinity of the second case-patient.

Early antemortem diagnosis of virus in an infected human is very important. Checking for virus in saliva eliminates the difficulty of tissue sampling from humans with suspected cases of rabies, and the sensitivity of hnRT-PCR makes it the technique of choice for detecting limited amounts of virus. Previous work showed that a 200-bp region of the N gene had only one nucleotide difference between them (4). Moreover, two samples from a region in western Mexico, isolated 30 years apart, were identical in sequence (4). Incorporation of the reference strains Pasteur and SAD B 19 into our phylogenetic tree indicated that the three human viruses we isolated belong to lyssavirus genotype 1.

References

(1.) Shimshony A. Veterinary public health in Israel. Revue Scientifique et Technique Office International Des Epizooties 1992;11:77-98.

(2.) Heaton PR, Johnstone P, McElhinnely M, Coweley R, O'Sullivan E, Whitby JE. Heminested PCR assay for detection of six genotypes of rabies and rabies related viruses. J Clin Microb 1997;35:2762-6.

(3.) Smith JS. Rabies virus. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 6th ed. Washington: American Society for Microbiology; 1995. p. 907-1003.

(4.) Smith JS, Seidel HD, Warner CK. Epidemiology and historical relationships among 87 rabies virus isolates determined by limited sequence analysis. J Infec Dis 1992;166:296-307.

Dan David,(*) Charles E. Rupprecht,([dagger]) Jean Smith,([dagger]) Itzhak Samina,(*) Shmuel Perl,(*) and Yehuda Strata*

(*)Kimron Veterinary Institute, Bet Dagan, Israel; and ([dagger])(Centers for Disease Control and Prevention, Atlanta, Georgia, USA