The outbreak of fever with altered sensorium began in late January 2001 and peaked in mid-February. No cases were reported after February 23 (Figure 1). All of the patients were residents of Siliguri, and some clustering of cases was observed around the Medinova Hospital, since the staff of this hospital resided in the area. Based on the case definition, 66 cases of encephalitis were identified, and the case-fatality ratio was ≈74%. The outcome of 61 cases was known; the remaining 5 patients were discharged from the hospital against medical advice. All patients were >15 years of age; the male-to-female ratio was 1.4:1. Forty-five (75%) of the 60 patients had a history of hospital exposure, i.e., they were members of the hospital staff or had attended to or visited patients in the hospital. The outbreak began at a single hospital, and cases were subsequently detected at 3 other hospitals. No definitive information about the possible index case exists. Five families had >1 case, but all of the persons affected had a history of hospital visits and had onset of illness 2 or 3 days apart from each other. The sequence of events is shown in Figure 2.

The patients initially had fever (100%), headache and myalgia (57%), vomiting (19%), altered sensorium (confusion to coma, 97%), respiratory symptoms (tachypnea to acute respiratory distress, 51%), and involuntary movements or convulsions (43%). No neck rigidity or cranial nerve involvement was observed in the 16 patients who were examined. Pupils were bilaterally dilated and reactive to light. Deep tendon reflexes were diminished or absent. Abnormal plantar reflexes (extensor plantar response) were elicited in 11 patients. Patients were normotensive at admission but became hypertensive before death. Death occurred within 1 week of onset of disease in 10 patients (62.5%), within 2 weeks in 5 (32.8%) patients, and on day 30 after onset for 2 patients.

Before the outbreak, the staff did not routinely use personal protective equipment or barrier nursing methods. Use of surgical masks was minimal on wards, except in the intensive-care units. Certain universal precautions, such as hand washing and use of gloves, were partially adhered to when staff were carrying out invasive procedures. Patients were housed on wards with >4 patients in a single room and could be visited or be attended to by their family and others. Patients did not wear masks on wards or when being transported for procedures (e.g., x-ray examination). Disposal of waste, collection of soiled linen, laundry, and cleaning of floors and other surfaces in the wards were carried out by personnel who did not follow infection control practices.

Once the outbreak of encephalitis was established, stringent infection control practices were introduced (Figure 1), including isolating patients in the Medical College Hospital, where 2 wards were established, one for suspected and the other for probable cases. Barrier nursing techniques were initiated, and housekeeping procedures and waste management were improved.

Cerebrospinal fluid was obtained from all patients. Analysis showed that the fluid in all cases was under pressure and clear with <5 lymphocytes/mm³ (normal range 0–5 cells/mm³). These samples were not available for further analysis.

Laboratory testing during and immediately after the outbreak did not identify a likely etiologic agent. Patient serum samples were tested for IgM antibodies to Japanese encephalitis, West Nile, dengue, and measles viruses as well as for Leptospira spp. Serum samples were also tested for IgG antibody to hantavirus. All serologic tests were negative, and no likely viral or bacterial agents were detected. All serum samples tested positive for IgG to measles virus.

Because NiV was identified as the cause of encephalitis outbreaks in Bangladesh, the Siliguri samples were tested for evidence of NiV infection. In all, 17 serum samples were available from 18 patients from Siliguri. All were tested for IgG and IgM antibodies to NiV by ELISA. The 6 urine samples collected from these 18 patients were tested for NiV RNA by RT-PCR, and aliquots were inoculated onto Vero E6 cells in an attempt to isolate NiV.

NiV-specific IgM and IgG were detected in 9 of 17 serum samples; 1 sample was positive for IgG and negative for IgM (Table). RT-PCR assays detected RNA from the N gene of NiV in 4 urine samples from NiV antibody–positive patients and in 1 urine sample from a NiV antibody–negative patient. RNA from the M gene was detected in 3 of these 5 samples (Table). No viral isolates were obtained from the 6 urine samples.

Sequence analysis confirmed that the PCR products were derived from NiV RNA (Figures 3 and 4). Partial N-gene sequences (159 nt) from 2 of 5 Siliguri samples were identical, and the other 3 sequences differed by no more than 1 nt, although unresolved sequence heterogeneity occurred at 2 positions (A or G) in 3 of the Siliguri N-gene sequences (Figure 3). Comparison of the Siliguri N-gene sequences to the N-gene sequences from NiV samples isolated in Bangladesh in 2004 and Malaysia in 1999 showed an overall level of nucleotide identity of 97.5%. Siliguri N-gene sequences were more closely related to the N-gene sequence from the Bangladesh isolate than to the sequences from the Malaysian isolates. Two of the Siliguri N-gene sequences were identical to the Bangladesh N-gene sequence.

Comparison of the partial M gene sequence amplified from the specimens from Siliguri to the M gene sequences from NiV isolated in Malaysia and Bangladesh (Figure 4) showed identity at 302 (94%) of 320 nt positions. Again, the Siliguri M gene sequences were more closely related to the M gene sequences from Bangladesh (99% identity) than to the sequences from Malaysia (94% identity).

This retrospective study provides evidence of NiV infection during a 2001 outbreak of febrile encephalitis in Siliguri. Nine of 18 of the patients tested had IgM and IgG antibodies; 1 had IgG antibodies only to NiV. Urine samples from 4 of these patients contained NiV RNA. One other patient had NiV RNA in the urine but lacked a detectable IgM and IgG response. In this case, the serum sample may have been obtained early in infection before antibodies to NiV were present. These laboratory results, along with the observation that the symptoms in the Siliguri patients were consistent with those described for patients during NiV outbreaks in Bangladesh and Malaysia (3–5,15–17), provide strong evidence that NiV caused the outbreak in Siliguri. Failure to detect evidence of NiV or NiV-specific antibodies in some patients may have been due to early sample collection or to inclusion of encephalitides of other causes because of the broad case definition. One patient was IgG-positive but had no detectable IgM, which suggests past infection by NiV. Unfortunately, because no case control and population-based studies were undertaken during this outbreak, interpreting this result is difficult.

The main reservoir for NiV is believed to be fruit bats of the genus Pteropus. NiV was isolated from fruit bats in Malaysia and Cambodia, and seropositive bats have been detected in other parts of Southeast Asia (7–10). In the Malaysian outbreak, commercially raised pigs were believed to be intermediate hosts. Presumably, the pigs were infected by virus shed from fruit bats and then transmitted the virus to humans. Although fruit bats with antibodies to NiV were captured in the outbreak areas of Bangladesh, no intermediate animal host was identified. In Bangladesh, NiV might have been transmitted to humans by direct contact with bats or indirectly by contact with material contaminated by bats. Person-to-person spread was also noted during the 2004 NiV outbreak in Faridpur, Bangladesh (4,5). The range of Pteropus giganteus, one of the flying foxes commonly found in south Asia (18), includes West Bengal. Therefore, the range of the proposed natural reservoir for NiV extends into northeastern India, and since the geographic features of West Bengal are similar to those of Bangladesh, environmental circumstances that favor transmission of NiV to humans would likely also be found in West Bengal.

Many of the epidemiologic features of the outbreak in Siliguri were similar to those of the recent NiV outbreaks in Bangladesh. In Bangladesh, no intermediate animal host was identified, whereas in Siliguri studies to detect an intermediate host were not conducted. In Siliguri, no samples were obtained from local wildlife or domestic animals. In both outbreaks, transmission occurred in healthcare settings through contact with infected persons. In Siliguri, the observation that only adults were affected supported the nosocomial transmission theory, as the number of children on the wards of hospitals was minimal. During infection, NiV is present in respiratory secretions and urine (19) and in both outbreaks, failure to use personal protective equipment probably contributed to the spread of the virus. Many of the patients had nasogastric tubes inserted or were intubated, procedures which made exposure to respiratory secretions more likely. Initiating adequate barrier nursing techniques helped to curtail further spread of infection.

Sequence analysis of PCR products confirmed NiV RNA. Unfortunately, no virus was isolated, and only limited sequence data could be obtained from the available clinical material. Analysis of the limited sequence data suggested that the NiV strains associated with the outbreak were more closely related to NiV isolated in Bangladesh than to NiV isolated in Malaysia. These data extend the previous observation that viruses circulating in different areas have unique genetic signatures (10,14) and suggest that these strains may have co-evolved within local natural reservoirs.

To our knowledge, NiV infection has not occurred previously in India; however, given the proximity of Siliguri to the areas of Bangladesh that experienced NiV outbreaks in 2001, 2002, and 2004, the outbreak is not surprising. Given the distribution of the locally abundant P. giganteus, the apparent natural reservoir of NiV in this area, outbreaks of NiV will likely continue to occur in Bangladesh and northern India. Establishing appropriate surveillance systems in these areas will be necessary so that NiV outbreaks can be detected quickly and appropriate control measures initiated. When NiV infections are suspected, infection control practices must be strengthened to avoid outbreaks in hospital settings, as apparently occurred in Siliguri.