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Nipah Virus Infection in Kozhikode, Kerala, South India, in 2018: Epidemiology of an Outbreak of an Emerging Disease
research-article
Author(s):
Bina Thomas ,
Priya Chandran ,
M. P. Lilabi ,
Biju George ,
C. P. Sivakumar ,
V. K. Jayadev ,
V. Bindu ,
R. S. Rajasi ,
Binsu Vijayan ,
Anu Mohandas ,
Nimin Hafeez
Publication date (Print):
Oct-Dec 2019
Publisher:
Wolters Kluwer - Medknow
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Abstract
Background:
An outbreak of the rare and highly pathogenic Nipah virus infection occurred in Kozhikode, Kerala, India, during May 2018.
Methodology:
Outbreak control activities included laboratory case confirmation and isolation. Contact surveillance was initiated and close contacts were home quarantined for the maximum incubation period of the disease. Field visits and verbal autopsy of the deaths were done to elicit the details of exposure.
Results:
Of the 18 confirmed cases, 16 succumbed (case fatality rate, 88.8%). The mean incubation period was 9 days. The transmission was person to person wherein the primary case served as a point source for 15 other cases including 2 health-care workers. The mean age of the affected cases was 41 years with male preponderance. More than 2600 contacts were under surveillance. The outbreak was contained within 3 weeks and declared closed by July the same year.
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Most cited references13
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Nipah Virus-associated Encephalitis Outbreak, Siliguri, India
Mandeep S. Chadha, James A. Comer, Luis Lowe … (2006)
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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Isolation of Nipah virus from Malaysian Island flying-foxes.
Kaw Chua, Chong Koh, Poh Hooi … (2002)
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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Foodborne Transmission of Nipah Virus, Bangladesh
Stephen Luby, Mahmudur Rahman, M. Jahangir Hossain … (2006)
Nipah virus was first recognized in a large human outbreak that affected 283 persons and caused 109 deaths in Malaysia in 1999 ( 1 , 2 ). The outbreak was preceded by a large Nipah outbreak among pigs ( 3 ). Contact with sick pigs was the primary risk factor for human Nipah virus infection ( 4 ). The porcine outbreak, in turn, was thought to be caused by transmission of Nipah virus from fruit bats to pigs. Antibodies against Nipah virus were identified in the 2 native Pteropus species ( 5 ), and the virus was subsequently isolated from pooled urine samples from a P. hypomelanus colony on Tioman Island, Malaysia ( 6 ). The most likely initiating event was that a fruit bat that was shedding Nipah virus in its saliva dropped a piece of partially eaten fruit into a pig sty, and 1 or more of the pigs became infected ( 1 , 7 ). Genetic characterization of the Nipah virus strains isolated from pigs in the Malaysia outbreak identified 2 strains, 1 of which was identified in humans, and 1 of which may have given rise to the other through genetic drift ( 8 ). These findings suggest that as few as 1 or 2 instances of spillover of Nipah virus from bats to pigs occurred. No further cases of Nipah virus have been reported in Malaysia since 1999. Four outbreaks of Nipah virus have been recognized in central and west Bangladesh from 2001 through 2004 ( 9 – 11 ) (Figure 1). Each outbreak occurred between January and May. Different outbreaks have been associated with different exposures. In the first outbreak in Meherpur in 2001, Nipah case-patients were significantly more likely to have had contact with a sick cow and contact with the secretions of an ill person than were controls ( 9 ). In Naogaon in 2003, case-patients were more likely than controls to have had contact with a herd of pigs that had passed through the area before the outbreak ( 12 ). In the 2004 outbreak in Goalando, Nipah case-patients were significantly more likely to have climbed trees and to have had contact with ill persons than were controls ( 13 ). In the 2004 outbreak in Faridpur, contact with ill persons was the primary risk for human Nipah disease ( 10 ). Figure 1 Location and dates of confirmed Nipah outbreaks in and near Bangladesh. Substantial data implicate flying foxes (Pteropus spp.) as the natural reservoir of Nipah virus. Investigations of Pteropus spp. in Malaysia, Cambodia, and Thailand have consistently identified antibodies against Nipah virus ( 5 , 14 – 16 ). It has been isolated from Pteropus spp. bats in Malaysia, Cambodia, and Thailand ( 6 , 15 , 16 ). P. giganteus is the only Pteropus species present in Bangladesh. In the Naogaon investigation, 2 of 19 P. giganteus specimens had antibody against Nipah virus. None of 31 other animals from various species had Nipah antibodies ( 9 ). Strains of Nipah virus isolated from affected persons in Bangladesh have substantial genetic diversity ( 17 ). The repeated outbreaks in Bangladesh and the genetic diversity of Nipah virus isolated from affected persons in Bangladesh suggest substantial diversity of the virus in the wildlife reservoir and repeated spillover of the virus from its reservoir to the human population. On January 11, 2005, government health workers in Tangail District reported that 8 previously healthy persons from Basail Upazila (subdistrict) had died within the preceding week from an illness characterized by fever and mental status changes. The Institute for Epidemiology Disease Control and Research (IEDCR) of the government of Bangladesh immediately launched an investigation and 5 days later invited the International Centre for Diarrheal Disease Research, Bangladesh (ICDDRB) to assist. The objectives of the investigation were to determine the cause of the outbreak, identify risk factors for development of illness, and develop strategies for prevention. Methods Case Identification From January 11 onward, government health workers at the Basail Health Center recorded the names and basic clinical information and collected a blood sample from all patients who sought treatment for fever, seizures, or mental status changes. They followed up each ill person at least once per week until he or she recovered. Government health authorities and hospital medical directors in surrounding areas were notified of the outbreak and encouraged to contact the IEDCR if any patients with symptoms of encephalitis from Tangail District sought treatment in a healthcare institution outside of the district. Basail Upazila is composed of 6 unions. Ultimately, the study team defined a case-patient with outbreak-associated encephalitis as a person who lived or traveled in Habla Union, Basail Upazila, Tangail District, Bangladesh, in whom a fever developed and who had new onset of seizures or altered mental status between December 15, 2004, and January 31, 2005. In-Depth Interviews An experienced anthropology team conducted in-depth interviews with the families of each case-patient. The objectives were 1) to explore potentially relevant exposures; 2) to assist in framing questions for the case-control questionnaire within the context of the activities, understanding, and language of local residents; and 3) to identify appropriate proxy respondents for each case-patient. Topics covered in the interviews included details on exposure to ill or dead persons; purchase and consumption of date palm sap; contact with animals, especially sick animals; availability and consumption of locally grown fruits, vegetables, and flowering plants; and presence and behavior of fruit bats in the area. The anthropology team interviewed collectors of date palm sap in detail about the context and process of date palm sap collection, preparation of the sap for consumption, and sales and distribution of the sap. Case-Control Study On the basis of the findings from the in-depth study, a closed-ended questionnaire was developed and translated into Bengali. Five interviewers with extensive prior experience in administering close-ended questionnaires were trained in a 2-day course on a standardized method to request informed consent and administer the questionnaire. The interviewers then pretested the questionnaire in the presence of supervisors on a sample population from a nearby village that was not included in the study. After final revisions, the interviewers administered the questionnaire to each case-patient or his or her proxy(ies). Controls were identified by visiting the next closest house to the case-patient, confirming that no one in the house met the case definition, identifying the household resident closest in age to the case-patient, and then seeking consent to administer the questionnaire. Only 1 control was enrolled per household. If the household resident closest in age to the case-patient declined to participate in the study, no other person in the household was sought as a control. This process was repeated at the next closest household until 3 controls were enrolled for each case-patient. Proxy respondents were identified for each case-patient who had died or was unable to respond. Proxy respondents included spouses, family members, and friends. Multiple proxy respondents were common; for example, a neighborhood playmate could be aware of food exposures that a parent might not be. Mapping We measured the location of key features in the outbreak by using global positioning system sensing. We then superimposed these locations on publicly available government maps. Laboratory Methods Whole blood specimens were transported on wet ice to the laboratory at ICDDRB, where they were centrifuged; the separated serum was stored at –70°C. Serum samples were shipped on dry ice to the Centers for Disease Control and Prevention (CDC) and tested with an immunoglobulin M (IgM) capture enzyme immunoassay (EIA) that detects Nipah IgM antibodies and an indirect EIA for Nipah IgG antibodies ( 18 ). Nipah (Malaysia prototype) virus antigen was used in both assays. Statistics We used odds ratios to estimate the association of each exposure with disease. To assess whether observations departed from what would be expected by chance, we used the χ2 test when expected cell sizes were >5 and the Fisher exact test when expected cell sizes were <5. We calculated exact mid-p 95% confidence limits around the odds ratio. We used an unmatched analysis because neighbors were chosen as controls to ensure that controls arose from the same population as case-patients and not to control for confounding factors. Indeed, all case-patients and controls lived within the same area. We enrolled persons closest in age, not to control confounding by age but rather to provide simple guidelines to the interviewers that would prevent the common tendency to disproportionately enroll heads of households. Only 1 exposure was significantly associated with illness in the initial analysis. To account for the lack of independence among the exposures of the 3 case-patients that occurred in the same household, we used a generalized estimating equations model with an exchangeable correlation matrix ( 19 ). Ethics The investigation team developed messages based on evidence from prior outbreaks on what steps family members could take to prevent person-to-person transmission of Nipah virus. During the outbreak, government health workers actively disseminated these messages. In addition, at the end of each in-depth interview carried out by the anthropology team, messages on steps to prevent person-to-person transmission of Nipah virus were directly communicated to case-patient families. Informed consent was requested of all study participants or their proxies. Because an emergency outbreak investigation was being conducted, the protocol was not reviewed by a human subjects committee. Results Government health workers identified 124 persons within the outbreak area in whom fever developed during January 2005. Of these, 12 persons met the outbreak-associated encephalitis case definition. Among case-patients, the most common accompanying symptom was lack of consciousness (Table 1). The patients' median age was 16 years (range 5–85 years); 7 (58%) were male. Eleven (92%) of the persons who met the case definition died. Death occurred a median of 5 days (range 4–9 days) after the first symptom of illness was reported. Table 1 Symptoms and signs of persons with outbreak-associated encephalitis, Habla Union, Bangladesh, January 2005 Symptom and signs No. (%) Fever 12 (100) Death 11 (92) Lack of consciousness 9 (75) Headache 5 (42) Vomiting 5 (42) Seizures 4 (33) Difficulty breathing 1 (8) The onset of illness for all of the case-patients occurred within 17 days, and all but the last case occurred after 10 days (Figure 2). All of the case-patients lived within 8 km of each other (Figure 3). Three cases occurred in a single household. Figure 2 Dates of Illness onset, encephalitis outbreak, Habla Union, Bangladesh. Figure 3 Outbreak area, Habla Union, Basail Upazila. Serum specimens were collected from 3 persons who met the outbreak-associated encephalitis case definition (Table 2). Two case-patients had IgM antibodies against Nipah virus by capture EIA. These 2 case-patients had blood collected 8 and 17 days after illness onset. The patient without detectable IgM antibody had his blood collected 2 days after illness onset. Both patients with IgM antibody also had Nipah IgG antibodies detected. Serum was also collected from 20 residents of the outbreak-affected community who had fever but did not meet the outbreak-associated encephalitis case definition. All 20 of these specimens were negative for Nipah IgM and IgG antibodies. Table 2 Serum Nipah capture antibody results, encephalitis outbreak, Habla Union, Bangladesh* Age, y Illness onset Date of serum collection Outcome Nipah IgM Nipah IgG 6 Dec 31, 2004 Jan 17, 2005 Survived + + 25 Jan 9, 2005 Jan 11, 2005 Died – – 12 Jan 16, 2005 Jan 24, 2005 Died + + *Ig, immunoglobulin. Case-Control Study Interviewers enrolled 11 patients who met the outbreak-associated encephalitis case definition and 33 matched controls. One patient was excluded because we could not identify proxy respondents with thorough knowledge of his exposures. Proxy respondents were used for all case-patient interviews and for 6 (17%) control interviews. The only exposure that was significantly associated with illness was drinking raw date palm sap (64% among cases versus 18% among controls, odds ratio [OR] 7.9, p = 0.01, Table 3). Of the 13 persons who reported consuming date palm sap, 11 knew the location where the sap had been harvested. Ten (91%) reported that the sap was harvested from a single village. None of the study participants were harvesters of date palm sap; none reported drinking the date palm sap directly from the collection container. Table 3 Bivariate analysis of risk factors for encephalitis Habla Union, Tangail District, Bangladesh, 2005 Risk factor Case-patients with this risk factor,
n = 11; no. (%) Controls with
this risk factor,
n = 33; no. (%) Odds ratio 95% Confidence limits* p value† Male sex 6 (55) 16 (49) 1.3 0.31– 5.4 0.73 Climbed trees 3 (27) 11(33) 0.8 0.14–3.4 1.0 Physical contact with living animal Pig 0 0 Undefined 1.0 Fruit bat 0 0 Undefined 1.0 Cow 5 (46) 21 (64) 0.48 0.11–2.0 0.31 Goat 2 (18) 6 (18) 1.00 0.12–5.8 1.0 Sheep 0 2 (6) 0 0, 11 1.0 Chicken 5 (46) 9 (27) 2.2 0.50–9.4 0.29 Duck 3 (27) 7 (21) 1.4 0.24–6.7 0.69 Cat 1 (9) 10 (30) 0.23 0.01–1.7 0.24 Physical contact with any sick animal 4 (36) 4 (12) 4.1 0.7–22 0.09 Physical contact with sick chicken 2 (18) 2 (6) 3.4 0.3–36 0.26 Killed a sick animal 0 1 (3) 0 0–57 1.0 Ate an animal that had been sick at the time it was killed 0 1 (3) 0 0–57 1.0 Drank raw date palm sap 7 (64) 6 (18) 7.9 1.6–38 0.01 Ate Banana 3 (27) 11 (33) 0.75 0.14–3.4 1.00 Papaya 1 (9) 7 (21) 0.37 0.01–2.9 0.66 Starfruit 2 (18) 8 (24) 0.7 0.09–3.8 1.0 Guava 5 (46) 14 (42) 1.1 0.27–4.6 1.0 Tamarind 1 (9) 3 (9) 1.0 0.03–11 1.0 Buroy 2 (18) 6 (18) 1.0 0.12–5.8 1.00 Traveled outside subdistrict 4 (36) 10 (30) 1.3 0.28–5.6 0.73 Touched someone with fever and altered mental status who later died 0 7 (21) 0.0 0.0–2.0 0.16 Been in the same room with someone with fever and altered mental status who later died 2 (18) 9 (27) 0.59 0.08–3.2 0.70 *Exact mid-p.
†All p values are 2-tailed. Fisher exact test used when expected cell size <5. A greater proportion of case-patients than controls reported physical contact with sick animals, although this difference may have been due to chance (36% vs. 12%, OR 4.1, p = 0.09). Two case-patients had contact with a sick hen, 1 with a sick cat, and 1 with a sick sheep. None of the 44 case-patients or controls reported physical contact with pigs or fruit bats, and none had eaten bats. Case-patients were no more likely than controls to have climbed trees or to have had contact with ill persons who later died. In the general estimated equation model that adjusted for the 3 cases clustered in the same household, drinking raw date palm sap was significantly associated with illness (adjusted OR 5.6, 95% confidence limits 1.7–7.9, p = 0.03). Qualitative Findings Date palm sap collectors explained that harvesting is a seasonal occupation that, in this region, begins in mid-December with the first cold night and continues through mid-February. At the beginning of the season, the bark is shaved off on 1 side of the tree (Phoenix sylvestris) near the top in a V shape, and a small hollow bamboo tap is placed at the base of the V. In the late afternoon, date palm sap collectors climb the tree, scrape the area where the bark is denuded so the sap can flow freely, and tie a 2- to 4-L clay pot under the tap. During the night, as the palm sap rises to the top of the tree, some leaks out where the bark is denuded, flows through the tap, and drips into the clay pot. Palm sap collectors climb the trees between 5:00 a.m. and 6:00 a.m. to gather the clay pots. The date palm sap from the individual clay pots typically contains 1–3 L of sap from a single tree; this sap is poured into a larger metal 22-L aluminum vessel with sap from several trees. Sellers will typically first walk to villages near where they collected the sap and either sell it door to door or from the road. If they still have some remaining, they will go to the market to sell it. Some buyers cook the sap to make molasses or special deserts; some is consumed fresh. Customers typically bring their own glass or jar, and the date palm sap seller dispenses the sap from the larger container. Fresh sap has to be sold early in the morning; otherwise, the sap will ferment and no longer taste sweet. The market price for fresh date palm sap was 8–10 taka (US $0.14–$0.17) per liter before 10:00 am and 4–5 taka (US $0.07–$0.09) after 10:00 am. Owners of date palm trees reported that they often hear bats at night. Owners viewed the fruit bats as a nuisance because they frequently drink the palm sap directly from the tap or the clay pot. Bat excrement is commonly found on the outside of the clay pot or floating in the sap. Occasionally dead bats are found floating in the pots. One of the Nipah case-patients who died was the son of a date palm sap collector. The collector harvested sap from his own date palm trees that grew in his household compound, as well from other trees in the area. The collector reported that he had recently heard bats near his trees at night and noted signs of bat excrement in and outside of the clay pot used to collect the sap. His son had been consuming date palm sap on a daily basis since the start of the season. Several days before the outbreak, the date palm sap collector sent a gift of fresh palm sap to his relatives living in a nearby homestead. Encephalitis developed in 3 members of the recipient household; 2 died. Discussion This outbreak was almost certainly caused by infection with Nipah virus. The tight clustering of cases in time and space suggests a single etiologic agent. The clinical signs and symptoms of fever, central nervous system involvement, and rapid progression to death are consistent with other Nipah outbreaks in Bangladesh. The outbreak occurred in the same region and during the same time of year as the 4 prior confirmed Nipah outbreaks in Bangladesh. Finally, 2 of 3 persons who met the outbreak-associated encephalitis case definition and were able to provide a serum specimen had IgM antibodies against Nipah virus. The single outbreak-associated specimen that was IgM negative was drawn from a patient on day 2 after symptom onset, so IgM antibodies may not have been present in sufficient quantity to be detected ( 20 ). Date palm sap was the likely vehicle of transmission for most of the Nipah virus infections in this outbreak. Drinking fresh, raw date palm sap was the sole exposure significantly associated with illness. Moreover, date palm sap is a biologically plausible vehicle. Although fruit bats uncommonly shed Nipah virus ( 5 , 21 ), when infected they can shed virus in both saliva and urine ( 5 , 15 , 16 ). Nipah virus can survive for days in fruit juice or flying-fox urine ( 22 ). Since date palm sap is sold and consumed within a few hours of collection, consumers could ingest infectious virus. This outbreak provides further evidence that Nipah virus infection in humans in Bangladesh is a seasonal disease that results from interaction between P. giganteus fruit bats and humans. This is the fifth Nipah outbreak in 5 years that has been identified in the same region (Figure 1). A Nipah outbreak also was confirmed in Siliguri, India, 15 km north of the border with Bangladesh in January 2001 ( 23 ). Each of these outbreaks occurred between January and May. P. giganteus is widely distributed throughout Bangladesh ( 24 ). The reason the outbreaks are occurring in this region at this time of year may be related to a seasonal increase in Nipah virus shedding among P. giganteus or to P. giganteus' attraction to particular natural or agricultural foods that are seasonally available in this region and bring the bats into proximity with humans. An important limitation of this investigation is that only 3 serum samples were available from case-patients. Thus, some persons included in the outbreak may not have had Nipah virus infection. Misclassifying non–case-patients as case-patients would bias the odds ratio toward null. A second limitation is that proxy interviews were required to obtain exposure information for all case-patient interviews. In addition, the same population was used for in-depth interviews and for follow-up quantitative questionnaires. Thus, information bias is possible. However, proxy respondents were independently identified by the qualitative research team to ensure that only persons who directly observed the case-patients' exposures were included. The standardized questionnaire used for the case-control study was pretested. The interviewers were unaware of the study hypothesis, and respondents were encouraged to answer to the best of their knowledge. Discrepancies were found between the in-depth interviews and the quantitative studies. Three proxy respondents who unambiguously reported consuming date palm sap during the initial in-depth qualitative interview reported no consumption during the follow-up close-ended interview. These 3 persons were relatives of a date palm sap collector. Their answers to the case-control questionnaire were used in the analysis. Thus, some study participants probably did realize what some of the investigation team's hypotheses were; however, this factor apparently biased the data against finding an association with date palm sap. Indeed, none of these sources of bias were likely to produce a spurious association between disease and raw date palm sap consumption. A third limitation is that Nipah virus was not isolated from date palm sap. Indeed, by the time the investigation implicated date palm sap, transmission of Nipah virus was no longer occurring in the area, and we did not collect date palm sap samples. However, the evidence favoring date palm juice as the vehicle for transmission of Nipah virus in this outbreak is stronger than for any alternative hypothesis. This study highlights the value of a diverse outbreak investigation team. Clinicians identified and cared for ill patients. Experts in qualitative research collaborated with the epidemiology team to understand potential routes of exposure and then used in-depth discussion with affected community residents to identify locally relevant exposures and frame the questions for the quantitative investigation. By conversing with residents of the affected area, qualitative investigators corrected outsiders' misconceptions about local exposures and were also able to quickly develop locally relevant messages to prevent secondary transmission. Laboratory investigators confirmed the cause of the outbreak. A close working relationship between government health workers and researchers permitted shared access to relevant information that provided government authorities with information on how to manage the outbreak and prevent further transmission. Investigation of different Nipah outbreaks in Bangladesh have identified different routes of transmission including climbing trees, contact with sick persons, and contact with sick animals ( 9 – 13 ). This investigation identifies another way that Nipah virus may be transmitted from P. giganteus to humans in Bangladesh. Fresh date palm sap is a national delicacy that is enjoyed by millions of Bangladeshis each winter. Apparently, most servings of fresh date palm sap are safe to drink. However, this investigation suggests that, at least occasionally, the sap contains a sufficient dose of Nipah virus to be fatal to humans. Further research to define how frequently this occurs is important. Persons who want to avoid ingesting Nipah virus from this route, should avoid drinking raw date palm sap. Low-cost interventions to restrict access of fruit bats to date palm taps and pots and that make fresh date palm sap safer should be developed and evaluated. In addition, continued research to better understand Nipah virus transmission between bats and the multiple pathways of human infection are important for developing sound prevention strategies.
Author and article information
Journal
Journal ID (nlm-ta): Indian J Community Med
Journal ID (iso-abbrev): Indian J Community Med
Journal ID (publisher-id): IJCM
Title:
Indian Journal of Community Medicine : Official Publication of Indian Association
of Preventive & Social Medicine
Publisher:
Wolters Kluwer - Medknow
(India
)
ISSN
(Print):
0970-0218
ISSN
(Electronic):
1998-3581
Publication date
(Print):
Oct-Dec 2019
Volume: 44
Issue: 4
Pages: 383-387
Affiliations
Author notes
Address for correspondence: Dr. Priya Chandran, Department of Community Medicine, Government Medical College,
Kozhikode - 673 008, Kerala, India. E-mail:
drpriyaclt@
123456gmail.com
Article
Publisher ID: IJCM-44-383DOI: 10.4103/ijcm.IJCM_198_19
PMC ID: 6881878
PubMed ID: 31802805
SO-VID: 2a1ae940-c539-42e4-b2c6-02724ac5088f
Copyright © Copyright: © 2019 Indian Journal of Community Medicine
License:
This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
History
Date
received
: 18
May
2019
Date
accepted
: 04
October
2019
Categories
Subject:
Original Article
ScienceOpen disciplines: Public health