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      Foodborne Transmission of Nipah Virus, Bangladesh

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          TOC summary line: Nipah virus was likely transmitted from fruit bats to humans by drinking fresh date palm sap.

          Abstract

          We investigated an outbreak of encephalitis in Tangail District, Bangladesh. We defined case-patients as persons from the outbreak area in whom fever developed with new onset of seizures or altered mental status from December 15, 2004, through January 31, 2005. Twelve persons met the definition; 11 (92%) died. Serum specimens were available from 3; 2 had immunoglobulin M antibodies against Nipah virus by capture enzyme immunoassay. We enrolled 11 case-patients and 33 neighborhood controls in a case-control study. The only exposure significantly associated with illness was drinking raw date palm sap (64% among case-patients vs. 18% among controls, odds ratio [OR] 7.9, p = 0.01). Fruit bats ( Pteropus giganteus) are a nuisance to date palm sap collectors because the bats drink from the clay pots used to collect the sap at night. This investigation suggests that Nipah virus was transmitted from P. giganteus to persons through drinking fresh date palm sap.

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          Nipah Virus-associated Encephalitis Outbreak, Siliguri, India

          During January and February of 2001, an outbreak of febrile illness with altered sensorium was observed in Siliguri, West Bengal, India. Siliguri is an important commercial center with a population of ≈500,000. It is near borders with China, Bangladesh, Nepal, and Sikkim. The outbreak occurred among hospitalized patients, family contacts of the patients, and medical staff of 4 hospitals. Japanese encephalitis, which is endemic in this area, was initially suspected, but the age group affected and the epidemiologic features suggested a different disease. Laboratory investigations conducted at the time of the outbreak failed to identify an infectious agent ( 1 ). Nipah virus (NiV), a recently emergent, zoonotic paramyxovirus ( 2 ), was implicated as the cause of a highly fatal (case-fatality ratio 38%–75%), febrile human encephalitis in Malaysia and Singapore in 1999 ( 1 ) and in Bangladesh during the winters of 2001, 2003, and 2004 ( 3 – 6 ). The natural reservoir of NiV is presumed to be fruit bats of the genus Pteropus. Evidence of NiV infection was detected in these bats in Malaysia, Bangladesh, and Cambodia ( 7 – 10 ). In the Malaysian outbreak, NiV was introduced into the pig population, and most of the human cases resulted from exposure to ill pigs ( 2 ). However, an intermediate animal host was not identified during the Bangladesh outbreaks, which suggests that the virus was transmitted either directly or indirectly from infected bats to humans. Human-to-human transmission of NiV was also documented during the outbreak in Faridpur, Bangladesh ( 4 , 5 ). Because the clinical manifestations of the cases in Siliguri were similar to those of NiV cases in Bangladesh ( 3 – 6 ), and because Siliguri is near affected areas in Bangladesh, a retrospective analysis of clinical samples was undertaken to determine if NiV was associated with the Siliguri outbreak. Methods Case Definition and Clinical Sample Collection A team of physicians and epidemiologists from the National Institute of Virology, Pune, India, along with local public health authorities, visited Siliguri. Investigations were conducted with the assistance of health authorities from West Bengal State and staff from the North Bengal Medical College Hospital. Medical records of patients who were hospitalized during the study period were examined, and their family members or caretakers were interviewed. Areas of the town in which cases occurred and the houses of patients who died were visited. Contacts and family members of patients who died were also interviewed. A broad working case definition was used for case detection. The case definition evolved over the course of the outbreak on the basis of information from case-patients admitted to different hospitals, review of the line list of patients, and interviews with contacts in the community. A suspected patient was one >15 years of age with acute onset of high-grade fever and headache. A probable patient was one >15 years of age who had high-grade fever and altered sensorium and encephalitis of unknown origin. Blood samples were available for 18 hospitalized patients and for 13 family contacts of patients who died 2–3 weeks earlier. Six urine samples (5 samples had corresponding serum samples) were also collected. Serologic Testing State health authorities conducted laboratory tests to rule out malaria and bacterial infections. Serologic tests to detect infection by Japanese encephalitis virus, West Nile virus, measles virus, dengue virus, Leptospira spp., and hantavirus were carried out at the National Institute of Virology. Serum samples were gamma-irradiated at the Centers for Disease Control and Prevention (CDC) before being tested for immunoglobulin G (IgG) and IgM antibodies to NiV and measles by enzyme-linked immunosorbent assay (ELISA), as previously described ( 2 , 11 , 12 ). Briefly, inactivated antigens for these ELISAs were prepared from gamma (60Co)-irradiated NiV-infected or mock-infected Vero E6 cells. Serum samples were tested in 4-fold dilutions from 1:100 to 1:6,400. Samples were considered positive for the IgM assay if the sum of the adjusted optical densities (OD) from all of the dilutions (OD from infected antigen well minus OD from the mock-infected antigen) was >0.75 through the entire dilution series, and the titer was >1:400. Similarly, samples were considered positive for IgG if the sum for the adjusted OD from all the dilutions was >0.90 through the entire dilution series, and the titer was >1:400. Detection of NiV by RT-PCR and Virus Isolation RNA was extracted from urine samples by using a Qiagen (Valencia, CA, USA) RNA extraction kit. Reverse transcription–polymerase chain reaction (RT-PCR) was performed with the SuperScript One-Step RT-PCR kit with Platinum Taq (Invitrogen, Carlsbad, CA, USA), as previously described ( 13 , 14 ). Reaction products were analyzed by agarose gel electrophoresis and ethidium bromide straining. PCR products were sequenced by using a cycle sequencing reaction with fluorescent dye terminators (Perkin-Elmer, Applied Biosystems Division, Foster City, CA, USA), and reaction products were analyzed with an ABI 3100 (Perkin-Elmer) automatic sequencer. Sequence data from multiple reactions were analyzed by using version 10.1 of the Genetics Computer Group Package (Accelrys, San Diego, CA, USA). Phylogenetic analyses were performed with PAUP version 4.01 (Sinauer Associates, Sunderland, MA, USA). Two sets of primers were used for RT-PCR reactions. Primer set NVNBF-4 (5´-GGAGTTATCAATCTAAGTTAG-3´) and NVNBR4 (5´-CATAGAGATGAGTGTAAAAGC-3´) amplified a 159-nucleotide (nt) region of the N gene of NiV. Primer set NVBMFC1 (5´-CAATGGAGCCAGACATCAAGAG-3´) and NVBMFR2 (5´-CGGAGAGTAGGAGTTCTAGAAG-3´) amplified a 320-nt region of the M gene. Virus isolation was attempted from the urine samples on Vero E6 cells as previously described ( 2 ). Results 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. Figure 1 Epidemic curve of outbreak of febrile encephalitis in Siliguri, India, January though February 2001, by number of hospital-associated and nonhospital-associated cases and deaths. The vertical, black arrow indicates when barrier methods were introduced for case management. Figure 2 Sequence of events in the Siliguri (SD) outbreak. 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. Table Serologic and PCR test results for clinical material from patients with encephalitis, Siliguri, India* Patient no. Days after onset of fever Serology† PCR (urine) IgM IgG N gene M gene 1 10 + + + NA 2 5 + + NA 3 9 + + + NA 4 10 + + NA NA 5 9 – – NA NA 6 10 – – + + 7 3 – – NA NA 8 7 – – NA NA 9 Unknown – – NA NA 10 1 – + NA NA 11 3 + + NA NA 12 5 + + + + 13 7 – – NA NA 14 6 + + NA NA 15 3 + + NA NA 16 8 – – NA NA 17 8 + + + + 18 2 NA† NA – – *PCR, polymerase chain reaction; IgM, immunoglobulin M; IgG, immunoglobulin G; NA, no sample available.
†Nipah virus–specific IgM or IgM by enzyme-linked immunosorbent assay. 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. Figure 3 Comparison of partial N-gene nucleotide sequences obtained from the Siliguri specimens (by patient number, see Table) to sequences obtained from Nipah virus isolates from Bangladesh (AY988601) and Malaysia (AF212302, AF376747). Letters indicate positions that differ from the reference sequence on the top line, Nipah-malaysia-1. Dots indicate nucleotide identity. R indicates A or G. Figure 4 A) Comparison of partial M-gene nucleotide sequences of Siliguri specimens to Nipah virus isolates from Bangladesh (Bangladesh-1:AY988601, Bangladesh-2:unpublished) and Malaysia (AF212302). Letters indicate positions that differ from the reference sequence on the top line, Nipah-Malaysia. Dots indicate nucleotide identity. B) Phylogenetic tree based on the sequence alignment shown in panel A. 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). Discussion 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.
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            Nipah Virus Encephalitis Reemergence, Bangladesh

            Nipah virus is a recently described zoonotic paramyxovirus that causes a highly fatal encephalitis in humans ( 1 , 2 ). The only previously reported outbreaks of Nipah virus occurred in Malaysia and Singapore from September 1998 to May 1999. In Malaysia, 265 encephalitis cases, primarily among pig farmers, and a 40% death rate were reported ( 3 – 5 ). Concurrent outbreaks of a respiratory and neurologic illness caused by Nipah virus occurred among pigs in the affected areas, and close contact with pigs, especially sick pigs, was the major risk factor for human infection ( 3 , 6 ). Person-to-person transmission of Nipah virus was not documented ( 7 ). In Singapore, 11 cases and 1 death were reported among abattoir workers who slaughtered pigs imported from affected areas of Malaysia ( 3 , 6 ). The outbreak was contained by the mass culling of >1 million pigs, and since then, no other outbreaks of Nipah virus have been reported in Malaysia ( 8 ). Subsequent investigations identified Pteropus bats as a possible natural host for Nipah virus ( 9 – 11 ). Pteropus bats are also believed to be the natural host for Hendra virus, a zoonotic paramyxovirus that is genetically related to Nipah virus and has been associated with fatal respiratory and neurologic illness among persons in Australia ( 12 ). In April and May 2001, a cluster of febrile neurologic illnesses with nine deaths was reported in a village in Meherpur District, Bangladesh. Preliminary investigations by the Bangladesh Ministry of Health and the World Health Organization (WHO) excluded a diagnosis of Japanese encephalitis, dengue fever, or malaria, but 2 of 42 serum specimens obtained from village residents in May 2001 showed reactive antibodies to Nipah virus antigen in tests performed at the U.S. Centers for Disease Control and Prevention (CDC). However, a comprehensive investigation of this outbreak was not conducted. In January 2003, a cluster of febrile illnesses with neurologic features and eight reported deaths occurred in adjoining villages in Naogaon District, ≈150 km from the village in Meherpur District. Similarities in the clinical manifestations observed among patients in Naogaon and Meherpur raised the question of whether the outbreaks were caused by the same agent. In March 2003, we conducted a detailed retrospective investigation to describe the outbreaks in Meherpur and Naoganon, characterize their clinical features, and determine the etiologic agents, presence of asymptomatic infection, risk factors for infection and disease, and possible animal reservoirs. Methods The field investigation took place March 6–16, 2003, and consisted of separate outbreak investigations in Meherpur and Naogaon districts, a cross-sectional study among healthcare workers in Meherpur Hospital, and an assessment of possible animal reservoirs in the outbreak regions. Because of the substantial time lapse between the outbreak period and the field investigation in Meherpur, approval for the Meherpur portion of the study was obtained from the appropriate ethical review committees. Informed consent was obtained from all participants, except for children 7 years of age. In both outbreak investigations, the surveys were conducted among residents >2 years of age, which consisted of an oral interview and collection of 10 mL of blood by venipuncture. Field research assistants used a standardized data collection instrument to collect information on demographics, symptoms of illness, exposure to ill patients, exposure to animals in the surrounding area, and other possible risk factors. When persons were deceased, an interview was conducted by proxy with a household member. Interviews were typically completed within 30 minutes. We attempted to verify clinical information on hospitalized patients from medical records, but records were either not found or contained incomplete clinical data. Meherpur Outbreak Investigation A population census was performed before the study by field research assistants, who visited each household and obtained information on the age and sex of each household member. Surveys took place in the villages of Chandpur (population 604), where persons who died or were hospitalized had resided, and Sishipara (population 237), an adjacent village located ≈1/2 km south of Chandpur. We defined the outbreak period as April 1 through May 31, 2001. Potential patients were identified from lists compiled by the initial WHO investigation and from self-reports of illness by village residents during the outbreak period. Household members were also surveyed as potential patients and assessed for clinically compatible illness or asymptomatic infection. To assess risk factors for infection, we enrolled controls by using simple random sampling of the numbered population census of remaining residents to select twice as many controls for each potential patient. Naogaon Outbreak Investigation Surveys were conducted in the adjacent villages of East Chalksita (population 529) and Biljoania (population 481); suspected deaths and hospitalizations caused by Nipah virus infection were reported from both villages. The outbreak period was defined as January 1–31, 2003. Because the scope of the outbreak was not well-defined, emphasis was given to case finding, which consisted of a household-to-household search by the field research assistants to identify potential cases. A sample of asymptomatic household members and other village residents, selected by simple random sampling from an available government population census, were surveyed for illness and serologic evidence of infection. Healthcare Worker Study A cross-sectional survey was performed at Meherpur District Hospital, where most of the ill residents from Chandpur were admitted with encephalitis symptoms. We interviewed and collected serum samples from healthcare workers whose job descriptions involved close contact with patients, such as physicians, nurses, orderlies, and nursing assistants. Information was obtained on demographics, symptoms of illness, the degree of contact with the patient, and type of barrier precaution used during patient care. Serum Sample Collection Blood specimens were centrifuged on site, transported on wet ice, and stored at –20°C. Serum samples were shipped frozen at –70°C to CDC and tested with an immunoglobulin (Ig) M capture enzyme immunoassay (EIA) for detection of Nipah/Hendra IgM antibodies and an indirect EIA for Nipah/Hendra IgG antibodies ( 13 ). Nipah (Malaysia prototype) virus antigen was used in both assays. Data Analysis Interview data were entered into Epi Info 6.04 (CDC, Atlanta, GA) and validated and analyzed by using SAS version 9 (SAS, Cary, NC). Based on serologic results, we defined a confirmed case as a case in a village resident with fever, headache, or altered level of consciousness within the specific outbreak period with antibodies reactive with Nipah antigen. A probable case was defined as a case in a resident with onset of fever plus headache or altered level of consciousness during the outbreak period who died before serum samples could be collected for testing. For the case-control study, univariate analysis was performed for each risk factor variable. Potential cases, including those in household members who did not have cases that met definition for confirmed or probable cases, were defined as noncases and analyzed together with the control group. Assessment of Animal Reservoirs In both districts, attempts were made to obtain representative samples from domestic and wild birds and mammals. Collections were based in part on relative abundance, suggestions of any history of ill animals indicated by village reports, and the likelihood for human contact. Domestic species were restrained manually, when possible, or sedated by the intramuscular administration of ketamine hydrochloride (≈5–10 mg/kg). Traps were set near village residences for small mammals, such as rodents and insectivores. Mist netting or hand collections for bats and birds occurred in and around homes, suspected flyways, roosts, such as abandoned buildings, and likely foraging areas, such as fruit plantations. In addition, samples were obtained from bats captured or killed by local villagers because they were suspected of feeding on fruit trees in nearby orchards. In Naogaon, villagers reported that a herd of pigs was in the vicinity of the village ≈2 weeks before the outbreak. Pigs owned by the same herder (but not from the same herd) were bled for serologic testing. Animal serum samples and tissue were shipped frozen to CDC, and all specimens were tested using an indirect EIA employing protein A/G conjugate for mammals and an antibird conjugate for avian species. Results Meherpur Outbreak Investigation In Meherpur, 13 residents, all from Chandpur (attack rate 2.1%), met the case definition: 4 had confirmed cases and 9 had probable cases. The outbreak, which spanned 1 month, began with the index patient, a 33-year-old farmer who had onset of symptoms on April 20, 2001 and died 6 days later. The outbreak ended with the last case which occurred in a 60-year-old woman, a neighbor of the index patient, with onset of symptoms on May 20 (Figure A). All nine persons with probable cases were hospitalized and died as a result of their illness before laboratory specimens could be collected (case-fatality rate = 69%). The average length of illness from onset to death was 6 days (range 3–10 days). All four persons with confirmed cases had IgG antibodies reactive to Nipah virus antigen (including two persons that had previous positive results from initial testing in May 2001). IgM antibodies were not detected in any of the specimens. Six (46%) of 13 patients were male; their ages were 4–60 years of age (median 38 years). A cluster of five cases occurred in persons from the same household as the index patient; the cluster consisted of the index patient's wife, son, brother, and sister. Eight separate households were affected, and 9 of the 13 persons with probable or confirmed cases were relatives of the index patient either by blood or marriage. Although patients with probable or confirmed cases lived in the western half of the village, no other obvious geographic clustering was noted; households with no cases were located in between those with cases. Figure Illness onset of probable and confirmed cases of encephalitis. A) Meherpur District, 2001. B) Naogaon District, 2003. Of 119 surveys completed in Meherpur, 96 (81%) were from Chandpur residents. Of these, 15 (16%) were initially identified as potential cases, 28 (29%) were their household members, and the remaining 53 (55%) were randomly selected as controls. All the patients came from the group identified as persons with potential cases in Chandpur; no patients were found among their household members or the randomly selected controls. The case-control analysis was restricted to Chandpur residents. With respect to baseline characteristics, patients (n = 13) did not differ from nonpatients (n = 83) by sex or occupation, but patients were older than nonpatients (mean age 40 vs. 27 years of age, p 90% of villagers reported that bats were frequently seen near their homes, patients and controls showed no differences in contact with bats or other animals, whether ill or well. Table 1 Risk factors for illness among patients and nonpatients, Chandpur village, Meherpur, Bangladesh, 2001 Characteristic Patients Nonpatients OR (95% CI)a Caring for or living with a person with a case 10/13 34/83 4·80 (1.23–18.8) Shared personal items 7/10 24/29 0·49 (0.09–2·56) Contact with secretions from a person with a case 7/9 11/29 5·73 (1.00–32·7) Any animal contact Chickens 13/13 83/83 Undefined Cows 13/13 68/83 6.11 (0.34–108.4) Dogs 12/13 78/83 0.76 (0.08–7.16) Goats 10/13 64/83 0.98 (0.25–3.97) Ducks 9/13 58/83 0.97 (0.27–3.45) Bats 3/13 36/83 0.39 (0.10–1.53) Sick animal contact Chicken 7/13 44/ 83 1.03 (0.32–3.34) Cow 8/13 14/83 7.89 (2.24–27.7) Dog 0/13 2/83 1.20 (0.05–26.5) Goat 5/13 23/83 1.63 (0.48–5.50) Duck 1/13 24/83 0.20 (0.03–1.66) aOR, odds ratio; CI, confidence interval. Naogaon Outbreak Investigation In Naogaon, 12 persons from both villages met the case definition (attack rate 1.1%): 4 cases were confirmed, and 8 were probable. All cases were found through the reports of initial deaths and hospitalizations; no patients were found among asymptomatic family members or randomly sampled village residents. The index case occurred in a 12-year-old boy with symptom onset on January 11, 2003, and the last case occurred in 12-year-old girl on January 28 (Figure B). All but one of the patients were hospitalized (one died before hospital admission), and all eight patients with probable cases died (case-fatality rate = 67%); mean period from onset of symptoms to death was 4 days (range 2–7 days). No diagnostic specimens were available from the deceased patients. Of the four confirmed patients, all had IgG antibodies and three had IgM antibodies reactive with Nipah virus antigen. Eight (67%) patients were male, and their ages were 4–42 years of age (median 12 years of age). Clustering of patients occurred in one household, in which the head of household became ill on January 14 and later died. Symptoms developed in his wife and three daughters 2 weeks later; only the two younger girls survived. Altogether, eight households were affected. As with Meherpur, no geographic clustering of affected households was seen, but in contrast to the outbreak in Meherpur, members of affected households had no blood relationship to those in other affected households. Clinical Features of Cases The typical clinical course was similar in both outbreaks and began with onset of fever, followed by headache and varying degrees of diminishing consciousness. In both outbreaks, a fever was found in all patients, followed by an altered level of consciousness in 22 (88%) and headache in 18 (72%) (Table 2). Cough (16 [65%]) and difficulty breathing (16 [65%]) were also common. Vomiting occurred in half of the patients, but seizures and diarrhea were uncommon. Although a significant difference was seen between patients with confirmed and probable cases only with regard to dyspnea (25% vs. 82%, p = 0.01), patients with probable cases (all of whom died) tended to have a higher proportion of all symptoms compared to patients with confirmed cases, all of whom survived. Table 2 Clinical characteristics of patients with probable and confirmed encephalitis cases, by case classification, Meherpur and Naogaon, Bangladesh, 2001 and 2003 Symptom Patients with Confirmed cases,
n (%) Probable cases,
n (%) p value Total patients
n (%) Fever 8 (100) 17 (100) 1.00 25 (100) Altered level of consciousness 6 (75) 16 (94) 0.17 22 (88) Headache 4 (50) 14 (82) 0.12 18 (72) Cough 3 (38) 13 (76) 0.08 16 (64) Dyspnea 2 (25) 14 (82) 0.01 16 (64) Vomiting 4 (50) 9 (53) 0.61 13 (52) Seizures 1 (13) 5 (29) 0.34 6 (24) Diarrhea 0 (0) 3 (18) 0.30 3 (12) Healthcare Workers Study A total of 46 healthcare workers (6 physicians, 20 nurses, 20 ward assistants) participated in the survey; 32 (70%) reported having contact with at least one of the encephalitis patients through the course of direct patient care. Of those who had direct patient contact, 12 (40%) used barrier precautions, such as gloves, masks, or gowns. One worker had unprotected mucous membrane contact with secretions of ill patients with encephalitis, and one reported a needlestick injury. Six workers reported an illness characterized by fever and headache in the period from the outbreak onset through June 30, 2001, but none reported mental status changes. None of the participating healthcare workers had antibodies reactive with Nipah virus antigens. Assessment of Animal Reservoirs No cluster of ill animals was observed or reported in either district. In Meherpur, serum samples were collected from two pigs and 31 bats, including 25 P. giganteus. None had antibodies reactive with Nipah virus antigens. In Naogaon, 50 animals were tested for evidence of Nipah-like virus infection: 10 birds, 4 pigs, 4 dogs, 2 shrews, 5 rodents, and 25 bats, including 19 P. giganteus. Antibodies reactive to Nipah virus antigen were detected in two P. giganteus adult females. Serum specimens from all other animals were negative. Discussion In these two outbreaks, antibodies reactive with Nipah virus antigen were found in seriously ill persons with encephalitis and antibodies were absent in asymptomatic persons or those without serious illness. These findings strongly suggest that Nipah, or a related virus, is the cause of both outbreaks. Nipah virus–associated illness has not been previously reported outside of Malaysia and Singapore. However, in contrast to the outbreaks in Malaysia, where animal illnesses were reported and close contact with pigs was strongly associated with Nipah virus infection in Bangladesh, no obvious zoonotic source has been identified. Pigs are infrequently found in Bangladesh, and no animal illnesses or die-offs in or around the affected villages were reported. Although case-control results indicated that patients were more likely to have contact with an ill cow, no such cow was available for testing, and the associations may have been due to chance. However, such potential risk factors need to be explored in future outbreak settings. Because antibodies reactive with Nipah virus were identified in local Pteropus bats, which reinforces previous findings, this genus may serve as the reservoir for this group of viruses ( 9 – 11 ). A possible explanation for acquisition of infection without an obvious domestic reservoir may be inadvertent direct contact with bats or bat secretions. Human-to-human transmission of Nipah virus was not shown in the Malaysia and Singapore outbreaks ( 7 ), but several findings from the Bangladesh outbreaks suggest that close contact may have resulted in transmission. In Meherpur and in Naogaon, clusters of cases occurred within family households, with dates of symptom onset occurring over a range of time. In Meherpur, relatives with close contact with patients became ill, and handling or exposure to secretions of patients was found to be a risk factor for illness. Nipah virus has been detected in respiratory secretions and urine of patients, which suggests that person-to-person transmission is possible ( 14 ). However, we cannot rule out the possibility that a common source within households and among relatives may have been responsible for infection. In contrast, we found no evidence for transmission of Nipah virus from patients to healthcare workers. Contact between secretions or blood of healthcare workers and patients was reported in only two instances, which is an insufficient number to assess transmissibility through these routes. However, the lack of symptoms and lack of detectable antibody to Nipah virus in all hospital staff we evaluated suggest that transmission from patients to healthcare workers is uncommon. The major clinical characteristics described in Bangladesh were generally similar to the characteristics described during the Nipah virus outbreaks in Malaysia and Singapore, with most persons having fever, headache, and an altered level of consciousness ( 2 ). In Bangladesh, a higher proportion of patients had an altered level of consciousness than those in Malaysia, although the results in our study relied on self-reporting, and objective descriptions of symptoms were not systematically documented. The absence of antibodies to Nipah/Hendra virus in asymptomatic persons suggests that subclinical infection did not occur or was an uncommon event, although subclinical infection has been previously documented ( 15 , 16 ). Among patients with probable or confirmed cases, patients in Naogaon tended to be younger (median age 12 years vs. 38 years of age) and to have a shorter interval from symptom onset to death (4 days vs. 6 days), compared to patients in the outbreak in Meherpur. Whether younger age is associated with a more fulminant course is uncertain, but the experience in Naogaon suggests that children appear to be as susceptible to infection as adults. We restricted our case definition to confirmed or probable cases and did not include suspected cases in classification, defined as a surviving resident with onset of fever plus headache or altered level of consciousness, without serologic evidence for Nipah virus infection. In Meherpur, five nonpatients would have had suspected cases; in Naogaon, 44 patients would have had suspected cases. In the absence of objective clinical and serologic findings, persons with suspected cases are more likely to be patients with false-positive test results. On the other hand, the sensitivity of the Nipah virus EIA is limited ( 13 ); determining whether suspected cases represented true Nipah virus infection versus another process (such as hysteria or a different clinical syndrome) is difficult. Another limitation, especially of the Meherpur investigation, was the difficulty of obtaining an accurate recollection of activities that took place almost 2 years before the study. Two features of this outbreak of Nipah virus encephalitis are distinct from previous outbreaks. A clear history of exposure to a specific species of animals was lacking, although bats in the region had serologic evidence of infection, and person-to-person spread may have been an important mode of transmission. Two independent clusters of cases suggest that this virus may sporadically infect humans. From January through April 2004, two new clusters of fatal Nipah virus encephalitis have been reported in Bangladesh. These outbreaks further underscore the need for enhancing regional surveillance for Nipah virus and clarifying transmission patterns. Also, increasing the capacity to conduct surveillance for new cases may add to our understanding of the disease and guide development of effective prevention strategies.
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              Isolation of Nipah virus from Malaysian Island flying-foxes.

              In late 1998, Nipah virus emerged in peninsular Malaysia and caused fatal disease in domestic pigs and humans and substantial economic loss to the local pig industry. Surveillance of wildlife species during the outbreak showed neutralizing antibodies to Nipah virus mainly in Island flying-foxes (Pteropus hypomelanus) and Malayan flying-foxes (Pteropus vampyrus) but no virus reactive with anti-Nipah virus antibodies was isolated. We adopted a novel approach of collecting urine from these Island flying-foxes and swabs of their partially eaten fruits. Three viral isolates (two from urine and one from a partially eaten fruit swab) that caused Nipah virus-like syncytial cytopathic effect in Vero cells and stained strongly with Nipah- and Hendra-specific antibodies were isolated. Molecular sequencing and analysis of the 11,200-nucleotide fragment representing the beginning of the nucleocapsid gene to the end of the glycoprotein gene of one isolate confirmed the isolate to be Nipah virus with a sequence deviation of five to six nucleotides from Nipah virus isolated from humans. The isolation of Nipah virus from the Island flying-fox corroborates the serological evidence that it is one of the natural hosts of the virus.
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                Author and article information

                Journal
                Emerg Infect Dis
                EID
                Emerging Infectious Diseases
                Centers for Disease Control and Prevention
                1080-6040
                1080-6059
                December 2006
                : 12
                : 12
                : 1888-1894
                Affiliations
                [* ]International Centre for Diarrheal Disease Research, Dhaka, Bangladesh;
                []Institute for Epidemiology Disease Control and Research, Dhaka, Bangladesh;
                []Centers for Disease Control and Prevention, Atlanta, Georgia, USA
                Author notes
                Address for correspondence: Stephen Luby, ICDDRB, GPO Box 128, Mohakhali, Dhaka 1212 Bangladesh; email: sluby@ 123456icddrb.org
                Article
                06-0732
                10.3201/eid1212.060732
                3291367
                17326940
                63842d83-1f89-444e-8273-6c7fb308776e
                History
                Categories
                Research

                Infectious disease & Microbiology
                bangladesh,encephalitis,nipah virus,research,chiroptera,epidemiology,disease outbreaks

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