Date Palm Sap Linked to Nipah Virus Outbreak in Bangladesh,2008

Abstract

Introduction:

We investigated a cluster of patients with encephalitis in the Manikgonj and Rajbari Districts of Bangladesh in February 2008 to determine the etiology and risk factors for disease.

Methods:

We classified persons as confirmed Nipah cases by the presence of immunoglobulin M antibodies against Nipah virus (NiV), or by the presence of NiV RNA or by isolation of NiV from cerebrospinal fluid or throat swabs who had onset of symptoms between February 6 and March 10, 2008. We classified persons as probable cases if they reported fever with convulsions or altered mental status, who resided in the outbreak areas during that period, and who died before serum samples were collected. For the case–control study, we compared both confirmed and probable Nipah case-patients to controls, who were free from illness during the reference period. We used motion-sensor-infrared cameras to observe bat's contact of date palm sap.

Results:

We identified four confirmed and six probable case-patients, nine (90%) of whom died. The median age of the cases was 10 years; eight were males. The outbreak occurred simultaneously in two communities that were 44 km apart and separated by a river. Drinking raw date palm sap 2–12 days before illness onset was the only risk factor most strongly associated with the illness (adjusted odds ratio 25, 95% confidence intervals 3.3–∞, p<0.001). Case-patients reported no history of physical contact with bats, though community members often reported seeing bats. Infrared camera photographs showed that Pteropus bats frequently visited date palm trees in those communities where sap was collected for human consumption.

Conclusion:

This is the second Nipah outbreak in Bangladesh where date palm sap has been implicated as the vehicle of transmission. Fresh date palm sap should not be drunk, unless effective steps have been taken to prevent bat access to the sap during collection.

Introduction

N ipah virus (NiV) causes encephalitis in humans and has a high fatality rate (Hossain et al. 2008, Luby et al. 2009). Species of fruit-bats in the Pteropus genus are the presumed natural reservoirs of NiV. NiV has been isolated and/or NiV RNA has been identified in bats in Malaysia (Chua et al. 2002, Rahman et al. 2010), Cambodia (Reynes et al. 2005), and Thailand (Wacharapluesadee et al. 2005). Researchers identified antibodies against NiV in Pteropus fruit-bats in Malaysia, India, and Bangladesh (Yob et al. 2001, Hsu et al. 2004, Epstein et al. 2008). There were seven recognized Nipah outbreaks in Bangladesh from 2001 to 2007 (Luby et al. 2009) (Fig. 1). Investigators implicated various routes of transmission in those outbreaks, including person-to-person transmission, drinking raw date palm sap, and contact with sick animals (Luby et al. 2006, 2009, Gurley et al. 2007).

FIG. 1.

Bangladesh map showing location of Nipah surveillance sites, previous Nipah outbreak areas, and February 2008 outbreak areas of Bangladesh.

Surveillance for human Nipah infection has been ongoing since 2006 in six hospitals serving communities in the northwestern districts of Bangladesh where previous Nipah outbreaks have been reported (Fig. 1). The surveillance has been conducted by the Institute of Epidemiology, Disease Control and Research (IEDCR), in collaboration with the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B).

On February 26, 2008, government health workers reported that one child from Manikgonj District died with illness characterized by fever, generalized body ache, cough, difficulty breathing, and mental status changes. Another two siblings from the same household were admitted to a subdistrict healthcare facility in Manikgonj District with similar symptoms. Both of them were referred to the District Hospital for more advanced medical care; one of them died on the way to the referral hospital and the other child was taken to a private hospital in Dhaka District, where the child died on the following day. On February 27, 2008, a Nipah Surveillance physician from the Rajbari District reported another cluster of encephalitis cases. Four patients from the same village were admitted to the Rajbari District Hospital; another patient from the same area died before reaching the hospital and another patient from the neighboring village died upon reaching the hospital. All of them presented with fever, generalized weakness, cough or respiratory distress, progressive mental status changes, and unconsciousness.

IEDCR initiated an investigation of both clusters in collaboration with ICDDR,B on the day they received the reports. The objectives of the investigation were to determine the cause of the outbreak, identify risk factors for illness, and develop strategies for prevention.

Materials and Methods

Study site and study population

The communities affected by the outbreak were located in Doulatpur Upazila (subdistrict) (population 155,674) of Manikgonj District and Baliakandi Upazila (population 186,562) of Rajbari District in central-western region of Bangladesh (Bangladesh Bureau of Statistics 2010). These sites are located 44 km apart and are separated by a river (Fig. 1). Date palm sap is widely harvested, sold, and consumed in both areas.

Case identification and sample collection

The investigation team sought for suspect cases who had fever and convulsions or altered mental status in the outbreak areas between February 6 and March 10, 2008 (Fig. 2). First, we visited the hospitals where the patients were treated. We identified the suspect case-patients and interviewed them or their proxy respondents. Then we searched for additional case-patients in the affected villages. We collected blood, cerebrospinal fluid (CSF), and throat swab samples from all the living suspect case-patients. The serum and CSF samples were aliquoted locally. The samples were transported to ICDDR,B in cold pack or in liquid nitrogen for storage in −70°C freezer. We classified persons as confirmed Nipah case-patients by the presence of immunoglobulin M (IgM) antibodies against NiV or by the presence of NiV RNA or by isolation of NiV from CSF or throat swabs. The probable cases were defined as suspect cases who died before sample collection or who had no IgM against NiV in serum collected within 8 days of onset of illness and who died before a follow-up serum sample could be obtained.

FIG. 2.

Date of illness onset from both clusters occurred over 6 days during February 2008, Manikgonj and Rajbari Districts, Bangladesh.

Case–control study

The investigation team returned to the outbreak communities to conduct a case–control study from March 5 to 10, 2008, to determine the risk factors for illness. We enrolled all confirmed and probable case-patients as cases. Individuals who lived in the same communities as the cases, and who were close in age and were free from any febrile illness with convulsions or altered mental status between February 6 and March 10, 2008, were eligible to be enrolled as controls. We identified controls by visiting the fourth closest house to the case-patient's, confirming that no one in the house met the case definition, and identifying the household resident closest in age to the case-patient. We enrolled only one control 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 four controls were enrolled for each case-patient.

Data collection

Trained interviewers collected information from cases and controls using a standardized structured questionnaire in Bengali language, based upon the questionnaires used in previous Nipah outbreak investigations in Bangladesh. We collected a detailed exposure history to previously identified risk factors for cases and respective controls for 1 month preceding the onset of illness of cases. For each case-patient who had died or was unable to respond and for each of the controls who were <10 years of age, we identified proxy respondents. Proxy respondents included spouses, family members, friends, and neighbors who were knowledgeable about the illness or the exposures of the case-patients and controls. We also conducted informal interviews with several date palm sap collectors and local community residents about the date palm sap collection procedure, recognition of bats in the areas, and possible contamination of date palm sap by bats. We used global positioning system to determine the location of the outbreak areas.

Laboratory analysis

Serum and CSF samples were tested for IgM and IgG antibodies against NiV using IgM-capture and indirect IgG enzyme immunoassay (Daniels et al. 2001). CSF and throat swab specimens from five patients were tested at the U.S. Centers for Disease Control and Prevention (CDC) laboratory for molecular detection and virus isolation of NiV. Real-time RT-polymerase chain reaction (rRT-PCR) was performed using the following primers that amplified a 112-nucleotide (nt) fragment spanning from position 538 to 660 in the NiV N gene: forward primer NVBNF2B-5′-CTGGTCTCTGCAGTTATCACCATCGA-3′, reverse primer NVBN593R 5′-ACGTACTTAGCCCATCTTCTAGTTTCA-3′, and probe NVBN542P 5′-CAGCTCCCGACACTGCCGAGGAT-3′, with the FAM dye incorporated at the 5′ end and a BHQ1 molecule at the 3′ end. PCR products were sequenced as previously described (Chadha et al. 2006), and were analyzed using Sequencher 4.10.1 software (Gene Codes).

Date palm sap evaluation

The field team also collected date palm sap early in the morning from both the outbreak areas from February 27 to March 5, 2008. Two separate aliquots for a sample were collected from a tree: one in viral transport medium and another in trizol. The sap specimens were stored in a cold box maintaining temperature around 2°C–8°C and transferred to liquid nitrogen within several hours and later stored in −70°C. The sap was tested at CDC for the presence of NiV RNA by rRT-PCR; the sap was also cultured for NiV.

Infrared camera observation

We identified seven date palm trees where sap was collected for the cases' consumption in Manikgonj and Rajbari District outbreak sites. To identify the possible contamination of date palm sap by bats' secretions and to understand bat sap contamination behavior, we mounted one motion-sensor-infrared camera focusing on date palm trees' shaved surface, sap stream, tap, and collection pot in each of the seven trees for a full night (5:00 PM to 6:00 AM).

Data analysis

We used exact logistic regression to estimate the univariate odds ratios (ORs) with 95% confidence intervals (CIs) between exposures and case status. We stratified on the case–control pairs to account for the matched design. We assessed for confounding by constructing a multivariate exact logistic regression model. We included all exposures during multivariate analysis that had (p<0.20) in the initial model and removed those exposures one at a time that were not significantly associated with case status. We performed all statistical analyses with STATA version 10.0.

Ethics

Interviewers obtained voluntary informed consent from all participants or proxies; for those <18 years of age, the team obtained individual assent as well as parental consent. This investigation was part of an emergency response to the outbreak, and so a complete human subjects review of all activities was not possible, but the Ethical Review Committee at ICDDR,B had previously reviewed and approved a general protocol for Nipah surveillance and response to outbreaks.

Results

The outbreak occurred in two adjoining Districts of Manikgonj and Rajbari over the same 6-day period in February 2008 (Fig. 2). We identified a total of 10 case-patients: 4 from Manikgonj and 6 from Rajbari Districts. Nine of them died (90%); one 12-year-old child from Rajbari survived. The median age of all case-patients was 10 years, and eight (80%) were males. All of the cases presented with fever, progressive altered mental status, and loss of consciousness. The mean duration from illness onset to death was 6 days (Table 1).

Table 1.

Profiles of the Case-Patients in February 2008 Nipah Outbreaks, Manikgonj and Rajbari Districts, Bangladesh

Characteristics	Total, n=10; no. (%)
Age
Mean (SD) in years	19 (17)
Median (range) in years	10 (7–55)
Male	8 (80)
Clinical features
Fever	10 (100)
Altered mental status	10 (100)
Loss of consciousness	10 (100)
Convulsions	0 (0)
Severe weakness	9 (90)
Muscle pain	6 (60)
Headache	5 (50)
Cough	5 (50)
Difficulty in breathing	4 (40)
Diarrhea	3 (30)
Joint pain	3 (30)
Vomiting	2 (20)
New onset of seizures	1 (10)
Died	9 (90)
Onset of illness to death (n=9)
Mean (range) in days	6 (1–10)
Category of cases
Confirmed Nipah	4 (40)
Probable Nipah	6 (60)

CSF specimens were available from five (50%) case-patients and serum specimens were available from six (60%). Four patients died before the investigation team could collect any specimen. The field team was able to collect a second set of serum specimens from three case-patients (30%) within 1–6 days of first sample collection. There were four (40%) confirmed and six (60%) probable case-patients. Table 2 shows the laboratory results of each case-patient.

Table 2.

Laboratory Results of Case-Patients in February 2008, Manikgonj and Rajbari Districts, Bangladesh

Case-patients				CSF					Serum-1					Serum-2					Throat swab
No	Site	Category	Sampling days after illness onset	IgM	IgG	Isolation	PCR	Sampling days after illness onset	IgM	IgG	Isolation	PCR	Sampling days after illness onset	IgM	IgG	Isolation	PCR	Sampling days after illness onset	IgM	IgG	Isolation	PCR
1	R	Confirmed	9	Neg	Neg	Neg	Neg	5	Pos	Neg	ND	ND	9	Pos	Pos	ND	ND	5	ND	ND	Neg	Neg
2	R	Confirmed	6	Neg	Neg	Neg	Neg	6	Neg	Neg	ND	ND	7	Pos	Neg	ND	ND	6	ND	ND	Neg	Pos
3	R	Confirmed	3	Neg	Neg	Neg	Neg	3	Neg	Neg	ND	ND	NA	NA	NA	ND	ND	3	ND	ND	Pos	Pos
4	M	Confirmed	6	Neg	Neg	Neg	Neg	6	Pos	Neg	NA	NA	12	NA	NA	ND	ND	6	ND	ND	Pos	Pos
5	R	Probable	8	Neg	Neg	Neg	Neg	8	Neg	Neg	ND	ND	NA	NA	NA	ND	ND	8	ND	ND	Neg	Neg
6	R	Probable	NA	NA	NA	NA	NA	4	Neg	Neg	ND	ND	NA	Neg	Neg	ND	ND	NA	NA	NA	NA	NA
7	R	Probable	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
8	M	Probable	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
9	M	Probable	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
10	M	Probable	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA

R, Rajbari; M, Manikgonj; IgM, immunoglobulin M; IgG, immunoglobulin G; PCR, polymerase chain reaction; Neg, negative; Pos, positive; NA, not available; ND, not done; CSF, cerebrospinal fluid.

Sequencing of the complete NiV nucleoprotein (N) ORF amplified from these two isolated viruses from conventional two-step RT-PCR indicated an identical match. Complete genome sequence analysis of the two isolates confirmed that the two viruses were identical (Lo et al. 2011). The N ORF sequence shared nt sequence identity at all but seven positions with NiV isolated from India in 2007 (accession FJ513078), and at all but 10 positions with NiV isolated from Bangladesh in 2004 (accession AY988601). The amino acid sequence of N ORF differs at only one position from the 2007 Indian (R211→Q) and the 2004 Bangladesh (D188→E) isolates (Table 3).

Table 3.

Nucleotide Differences Between Nipah Virus Isolated from Bangladesh (2004, 2008) and India (2007)

Nipah virus isolate ID	Nucleotide position
							1	1	1	1	1	1	1
	2	5	5	6	6	6	0	1	2	3	4	5	5
	7	4	6	3	3	5	7	1	6	1	9	0	3
	0	9	4	2	3	4	7	9	9	1	7	0	3
NIVBGD2004RAJBARI1 AY988601	T	C	T	A	A	A	T	G	A	A	C	A	A
NIVBGD2008MANIKGONJ	C	A	G	.	G	G	.	A	G	G	T	.	G
NIVBGD2008RAJBARI	C	A	G	.	G	G	.	A	G	G	T	.	G
NIVIND2007NADIA FJ513078	C	A	G	G	.	.	C	A	G	.	T	G	.

NIVBGD2004RAJBARI1 AY988601 serves as consensus sequence by which to compare the others.

“.” indicates nucleotide identity with consensus sequence.

We enrolled 40 controls for the 10 case-patients in the case–control study. All case-patient interviews were conducted by proxies as nine patients had died before the investigation began, and the final child was still recovering from the illness and was unable to communicate with us. We also identified proxies for 19 (48%) of the controls who were <10 years old. None of the selected cases, controls, or proxies refused to participate in the study.

In both outbreak areas, all of the case-patients drank fresh raw date palm sap 2–12 days before onset of their illness compared with 10 (25%) controls who consumed fresh date palm sap during the period of investigation (p<0.001). Household members of case-patients were more frequently involved in date palm sap harvesting than household members of controls (30% vs. 3%, p<0.05). None of the case-patients had any history of physical contact with bats, although people from the community often reported seeing bats in the tapped date palm trees during sap collection. A greater proportion of case-patients than controls reported physical contact with apparently healthy live cats (60% among cases vs. 10% among controls, p<0.05). None of the case-patients had physical contact with sick animals, nor did they eat any sick animals. Two cases slept in the same room (20% among cases vs. 30% among controls, p>0.05) and one case had physical contact (10% among cases vs. 0% among controls, p>0.05) with other case-patients 2–3 days before their illness onset (Table 4).

Table 4.

Bivariate Analysis of Exposures for Nipah Virus Infection in February 2008, Manikgonj and Rajbari Districts, Bangladesh

Exposures	Case patients with this exposure, n=10; no. (%)	Control patients with this exposure, n=40; no. (%)	p-Value	Odds ratio	95% confidence intervals
Drank raw date palm sap	10 (100)	10 (25)	0.000	38	5.4–∞
Date sap
Given	6 (60)	4 (10)	0.656	2.2	0.3–19
Purchased
From local vendor	0 (0)	1 (3)	0.429	Undefined	Undefined
From any house	1 (10)	2 (5)	0.486	1.0	0–39
From any other sources	3 (30)	0 (0)	0.143	1.0	0-∞
Collected from tree	0 (0)	3 (8)	0.211	0.2	0–2.1
Date sap harvesting
Self	0 (0)	3 (8)	1.000	1.1	0–10
Household members	3 (30)	1 (3)	0.022	15	1.0–860
Climbed trees
Fruit trees	5 (50)	9 (23)	0.099	6.2	0.5–351
Date palm trees	0 (0)	4 (10)	0.549	0.4	0–4.3
Physical contact with living animals
Cow	4 (40)	17 (43)	1.000	0.9	0.2–4.5
Cat	6 (60)	4 (10)	0.002	11	1.9–84
Physical contact with sick animals
Cow	0 (0)	1 (3)	1.000	3.9	0–152
Chicken	0 (0)	1 (3)	1.000	4.1	0–159
Physical contact with dead animals (chicken)	1 (10)	0 (0)	0.200	4.2	0.1–∞
Ate sick animals	0 (0)	3 (8)	1.000	1.0	0–11
Ate dropped fruit
Banana	0 (0)	1 (3)	1.000	11	0–429
Local plum (Boroi)	5 (50)	24 (60)	0.389	0.5	0.1–3.9
Papaya	0 (0)	4 (10)	1.000	Undefined	Undefined
Carambola (Kamranga)	1 (10)	1 (3)	0.250	2.0	0.1–∞
Guava	0 (0)	13 (33)	0.435	0.3	0–13
Tamarind	4 (40)	11 (28)	1.000	1.0	0–66
Custard apple	1 (10)	0 (0)	0.333	Undefined	Undefined
Traveled outside subdistrict	1 (10)	6 (15)	1.000	0.6	0–6.3
Touched persons with fever and altered mental status who died later	1 (10)	0 (0)	0.184	4.1	0.1–∞
Been in the same room with persons with fever and altered mental status who died later	2 (20)	3 (8)	0.258	2.9	0.2–30

Bolded type indicate significant results.

In the Manikgonj cluster, three children from one family drank raw date palm sap, collected by their father, a local gachi or date palm sap collector. They drank the sap early in the morning on February 11 for the last time and subsequently two of them developed illness on February 20 and the third on February 23. The fourth child, who developed illness on February 21, was a resident of Dhaka District but visited his grandmother's house on February 6 for 12 days. His grandmother, a neighbor of the date palm sap collector, purchased raw date palm sap from him and served it to her grandson the same day the other children drank the sap.

In the Rajbari cluster, three members from one family (mother and her two children) shared date palm sap purchased from the neighborhood date palm sap collector with two other neighborhood residents (brother-in-law and nephew of that mother) on February 18; all five subsequently developed illness. A salesman who resided nearly 5 km away from those households visited the village that morning and also drank the sap offered to him. He also died with the similar symptoms to the other four cases in Rajbari.

The median incubation period from intake of raw date palm sap to the onset of illness was 10 days (range: 9–12 days) in Manikgonj and 4 days (range: 2–7 days) in Rajbari District. All of the cases had consumed about 100 mL of date palm sap. All of the cases consumed the sap before 9 AM.

Cases were more likely to be exposed to three risk factors than controls in the initial bivariate analysis. However, in the multivariate analysis, only a single risk factor, drinking raw date palm sap, was significantly and independently associated with the illness. Nipah cases were 25 times more likely than controls to have consumed raw date palm sap (adjusted ORs 25, 95% CIs 3.3–∞, p<0.001) in the preceding month. Physical contact with a living cat was also associated with illness in univariate analysis (ORs 11, 95% CIs 1.9–84, p=0.002), but was not statistically significant in the multivariate analysis (adjusted ORs 9.2, 95% CIs 0.6–675).

Infrared photographs showed that bats frequently visited date palm trees during sap collection. During seven nights of camera observation, 104 bats visits were photographed around the date palm tree's sap producing area (mean: 14.9 visits per tree per night standard deviation [SD] 30.1) with 47 visits to the shaved surface (mean: 6.7 visits per tree per night SD 13.1). Bats were seen to lick date palm sap 59 times during the observation, and almost half of them (49%) were Pteropus bats. The fresh date palm sap samples (15 samples collected from 7 trees for 8 consecutive days), collected at least 9 days after consumption by the last case of this outbreak, were negative for NiV RNA by PCR and virus isolation.

Discussion

This outbreak, involving the death of nine people in two communities separated by a river in Manikgonj and Rajbari Districts over a 6-day period in February 2008, was almost certainly caused by NiV infection. The presenting clinical signs and symptoms of the case-patients were fever, central nervous system involvement, and rapid progression to death, which are consistent with other Nipah outbreaks in Bangladesh (Hossain et al. 2008). Four of the case-patients from both communities had laboratory evidence of Nipah infection; tight clustering in space and time of all case-patients, including those who were not laboratory confirmed and drinking of raw date palm sap on the same day from the same pot, which is a known risk factor for NiV infection, supports the hypothesis that the probable cases also had Nipah infection. This is the second Nipah outbreak where date palm sap has been implicated as the exposure most strongly associated with the illness.

Pteropus bats are the presumed reservoir of NiV (Chua et al. 2002, Rahman et al. 2010). They shed the virus in both saliva and urine (Reynes et al. 2005, Middleton et al. 2007); Pteropus bats were observed to be licking the raw date palm sap collected in the outbreak areas. Indeed, in contrast to an earlier infrared camera study in Bangladesh, where only 5% of the bats that contacted date palm sap were Pteropus bats (Khan et al. 2011), when we evaluated the trees those were the sources of date palm sap consumed by cases in this outbreak, 49% of the bats that visited the tress and contacted date palm sap were Pteropus bats. There is evidence of survival of NiV in mango flesh, mango juice, pawpaw juice, and lychee juice for up to 3 days, depending upon the pH of the juice (Fogarty et al. 2008). Moreover, the half-life of NiV in bats' urine, with pH adjusted to 7 at a temperature up to 22°C, is 18 h (Fogarty et al. 2008). The pH of date palm sap is 7.2 (Aidoo et al. 2006), suggesting that survival of NiV in date palm sap may be similar. In Bangladesh, winter is the peak collection period of date palm sap and this Nipah outbreak corresponds with the seasonality of all previously reported Nipah outbreaks in Bangladesh. In winter, the temperature remains between 15°C and 28°C (Bangladesh Meteorological Department 2008) and this low temperature might extend the survival of NiV in bat secretions or in sap (Fogarty et al. 2008). As date palm sap is usually collected early in the morning (5 AM–7 AM) and all of the cases in this outbreak drank the raw date palm sap before 9 AM, NiV apparently survived until that time.

The date palm sap samples that were evaluated for the presence of NiV were collected 24 days after the onset of illness of the last identified case in the outbreak. The median incubation period of NiV is 9 days (Hossain et al. 2008). The absence of NiV in the sap 2 weeks after the putative transmission event suggests that date palm sap is only intermittently contaminated, a pattern of contamination that is consistent with the observed intermittent outbreaks in Bangladesh.

The distance between the two areas (44 km) was within the 50 km foraging ranges for the Pteropus bat (Kunz and Jones 2000). The genetic sequences of the isolated viruses from the two sites were identical in contrast to substantial diversity in NiV isolates noted previously from different outbreaks in Bangladesh (Harcourt et al. 2005). While we do not know if the same bat or the same colony of bats contaminated date palm sap at these two sites, the near simultaneous occurrence of these uncommon outbreaks by an identical strain of NiV and the similar pattern of transmission suggests that they resulted from the same underlying process.

Although the association between cats and Nipah infection was not statistically significant in the multivariate model, cats are susceptible to infection with NiV and when infected can shed virus in their saliva (Middleton et al. 2002). Other domestic animals, including pigs and cattle, were associated with Nipah illness in earlier outbreaks in Bangladesh (Luby et al. 2009). The role of domestic animals in transmission of NiV is an important area for continued research.

Limitations include that we could not test samples from four cases as they died before specimens could be collected, which might be misclassified as cases in our study, but all of these cases were previously healthy people whose symptoms were consistent with confirmed Nipah cases. Moreover, we could not collect follow-up samples from the patients, who were negative in the samples collected 3, 4, and 8 days of illness onset. However, in previously investigated outbreaks, IgM against NiV was present in the follow-up samples collected 2 or more weeks after illness onset among 56% of the Nipah cases, who did not have detectable IgM against NiV detected from earlier specimens (Hossain et al. 2008). So, it is likely that the probable cases in this outbreak also had Nipah illness. During the outbreak periods, the Government of Bangladesh conducted local awareness raising activities to notify the community about Nipah-like symptoms and to avoid drinking raw date palm sap, eating partially eaten fruits, and having contact with bats. This might have sensitized our study population regarding their response, but awareness regarding the risk factors possibly motivated study subjects and their proxies to recall their exposure history more elaborately, rather than encouraging them to hide those exposures. So, it is unlikely that the awareness raising activities affected the results of our study.

There is evidence of recurrent fatal outbreaks from 2001 through 2007 by the transmission of NiV from its fruit-bat reservoirs to humans in Bangladesh (Luby et al. 2009). The present investigation also suggests that date palm sap is an important pathway for this transmission. Drinking fresh date palm sap was the most strongly associated risk factor among the exposures investigated for this outbreak of human NiV infection in this study. The outbreak ended following local warning against drinking fresh date palm sap from the Government of Bangladesh. To prevent this illness, date palm sap should not be drunk fresh unless effective steps have been taken to prevent bat access to the sap during collection. We are working with local date palm sap collectors to develop socially acceptable low cost technologies to prevent bats' access to the date palm sap producing parts of the tree. Studies in Bangladesh involving local date palm sap collectors suggest that using a bamboo-skirt to cover the shaved part of the date palm tree and sap collection pot might be a practical, affordable method to prevent bats' access to the date palm sap (Nahar et al. 2008, Khan et al. 2011). Drinking raw date palm sap is a long-practiced tradition in Bangladesh, so public health recommendations to avoid drinking fresh date palm sap are unlikely to be universally followed. Research to identify culturally acceptable approaches to produce safe date palm sap may provide an additional lifesaving prevention strategy.

Footnotes

Acknowledgments

This research activity was funded by the Government of Bangladesh and the U.S. Centers for Disease Control and Prevention. ICDDR,B acknowledges with gratitude the commitment of Government of Bangladesh and the U.S. Centers for Disease Control and Prevention to ICDDR,B's research efforts. We extend thanks to Dr. Imtiaz Ashraf Chowdhury and Dr. Sayma Afroze of IEDCR. Thanks also to Dr. Abu Shahid from Nipah surveillance team at Rajbari District, Dr. Khondker Mahbuba Jamil from IEDCR for laboratory support, Dr. Shahed Sazzad and Mr. Dawlat from ICDDR,B for data collection, and Ms. Dorothy Southern for her support in preparing the article. We also thank the two communities for participating in our study.

Disclosure Statement

No competing financial interests exist for any of the authors.

Disclaimer Statement

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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