Date palm sap linked to Nipah virus outbreak in Bangladesh, 2008.
ABSTRACT 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.
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.
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.
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.
- SourceAvailable from: Joseph Prescott[Show abstract] [Hide abstract]
ABSTRACT: Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×108 TCID50 of Nipah virus resulted in neurological disease in 5 out of 8 hamsters, indicating that food-borne transmission of Nipah virus can indeed occur. In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing and mitigating future outbreaks.PLoS Pathogens 03/2014; 10(3):e1004001. · 8.06 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Bat-borne viruses can pose a serious threat to human health, with examples including Nipah virus (NiV) in Bangladesh and Malaysia, and Marburg virus (MARV) in Africa. To date, significant human outbreaks of such viruses have not been reported in the European Union (EU). However, EU countries have strong historical links with many of the countries where NiV and MARV are present and a corresponding high volume of commercial trade and human travel, which poses a potential risk of introduction of these viruses into the EU. In assessing the risks of introduction of these bat-borne zoonotic viruses to the EU, it is important to consider the location and range of bat species known to be susceptible to infection, together with the virus prevalence, seasonality of viral pulses, duration of infection and titre of virus in different bat tissues. In this paper, we review the current scientific knowledge of all these factors, in relation to the introduction of NiV and MARV into the EU.Viruses 05/2014; 6(5):2084-121. · 3.28 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Henipaviruses are associated with pteropodid reservoir hosts. The glycoproteins G and F of an African henipavirus (strain M74) have been reported to induce syncytium formation in kidney cells derived from a Hypsignathus monstrosus bat (HypNi/1.1), but not in the non-chiropteran cells BHK-21 and Vero76. Here we show that syncytia are also induced in two other pteropodid cell lines from Hypsignathus monstrosus and Eidolon helvum bats upon co-expression of the M74 glycoproteins. The G protein was transported to the surface of transfected chiropteran cells, whereas surface expression in the non-chiropteran cells was detectable only in a fraction of cells. By contrast, the G protein of Nipah virus is transported efficiently to the surface of both chiropteran and non-chiropteran cells. Even in chiropteran cells, M74-G was predominantly expressed in the ER as indicated by colocalization with marker proteins. This result is consistent with the finding that all N-glycans of the M74-G proteins are of the mannose-rich type as indicated by sensitivity to endo H treatment. These data indicate that the surface transport of M74-G is impaired in available cell culture systems with larger amounts of viral glycoprotein present on chiropteran cells. The restricted surface expression of M74-G explains the reduced fusion activity of the glycoproteins of the African henipavirus. Our results suggest strategies for the isolation of infectious viruses which is necessary to assess the risk of zoonotic virus transmission IMPORTANCE: Henipaviruses are highly pathogenic zoonotic viruses associated with pteropodid bat hosts. Whether the recently described African bat henipaviruses have a similarly high zoonotic potential as their Asian and Australian relatives is unknown. We show that surface expression of the attachment protein G of an African henipavirus, M74, is restricted in comparison to the G protein of the highly pathogenic Nipah virus. Transport to the cell surface is more restricted in non-chiropteran cells than it is in chiropteran cells explaining the differential fusion activity of the M74 surface proteins in these cells. Our results imply that surface expression of viral glycoproteins may serve as a major marker to assess the zoonotic risk of emerging henipaviruses.Journal of Virology 08/2014; · 4.65 Impact Factor
Date Palm Sap Linked to Nipah Virus Outbreak
in Bangladesh, 2008
Muhammad Aziz Rahman,1,2Mohammad Jahangir Hossain,1Sharmin Sultana,2Nusrat Homaira,1,2
Salah Uddin Khan,1Mahmudur Rahman,2Emily S. Gurley,1Pierre E. Rollin,3Michael K. Lo,3
James A. Comer,3Luis Lowe,3Paul A. Rota,3Thomas G. Ksiazek,3,4Eben Kenah,5
Yushuf Sharker,1and Stephen P. Luby1,3
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
44km 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–N,
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.
Key Words: Bats—Epidemiology—Surveillance—Transmission—Zoonosis.
2009). Species of fruit-bats in the Pteropus genus are the pre-
sumed natural reservoirs of NiV. NiV has been isolated and/
or NiV RNA has been identified in bats in Malaysia (Chua
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.
ipah virus (NiV) causes encephalitis in humans and
has a high fatality rate (Hossain et al. 2008, Luby 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 transmis-
sion in those outbreaks, including person-to-person trans-
mission, drinking raw date palm sap, and contact with sick
animals (Luby et al. 2006, 2009, Gurley et al. 2007).
Surveillance for human Nipah infection has been ongoing
since 2006 in six hospitals serving communities in the north-
western districts of Bangladesh where previous Nipah out-
breaks have been reported (Fig. 1). The surveillance has
been conducted by the Institute of Epidemiology, Disease
1International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh.
2Institute of Epidemiology, Disease Control and Research, Dhaka, Bangladesh.
3Centers for Disease Control and Prevention, Atlanta, Georgia.
4Galveston National Laboratory, Department of Pathology, University of Texas Medical Branch, Galveston, Texas.
5Department of Biostatistics, University of Washington Seattle, Seattle, Washington.
VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 12, Number 1, 2012
ª Mary Ann Liebert, Inc.
Control and Research (IEDCR), in collaboration with the
International Centre for Diarrhoeal Disease Research, Ban-
that one child from Manikgonj District died with illness
characterized by fever, generalized body ache, cough, diffi-
culty breathing, and mental status changes. Another two
siblings from the same household were admitted to a sub-
district 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
admittedtotheRajbariDistrict Hospital;anotherpatient 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 col-
laboration with ICDDR,B on the day they received the re-
ports. 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 Man-
ikgonj 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
44km 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, ce-
rebrospinal fluid (CSF), and throat swab samples from all the
aliquoted locally. The samples were transported to ICDDR,B
in cold pack or in liquid nitrogen for storage in -70?C freezer.
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.
The investigation team returned to the outbreak commu-
nities 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
veillance sites, previous Nipah outbreak areas, and February
2008 outbreak areas of Bangladesh.
Bangladesh map showing location of Nipah sur-
over 6 days during February 2008, Manikgonj and Rajbari
Date of illness onset from both clusters occurred
66RAHMAN ET AL.
repeated atthenext closesthouseholduntil four controlswere
enrolled for each case-patient.
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 re-
spondents. 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 lo-
cation of the outbreak areas.
Serum and CSF samples were tested for IgM and IgG an-
tibodies against NiV using IgM-capture and indirect IgG en-
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)
CACCATCGA-3¢, reverse primer NVBN593R 5¢-ACGTACT
TAGCCCATCTTCTAGTTTCA-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 col-
lected from a tree: one in viral transport medium and another
in trizol. The sap specimens were stored in a cold box main-
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 col-
lected for the cases’ consumption in Manikgonj and Rajbari
District outbreak sites. To identify the possible contamination
of datepalm sapbybats’ secretions and tounderstand 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).
We used exact logistic regression to estimate the univariate
odds ratios (ORs) with 95% confidence intervals (CIs) be-
tween 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 multi-
variate analysis that had (p<0.20) in the initial model and
removed those exposures one at a time that were not signifi-
cantly associated withcase status. We performed all statistical
analyses with STATA version 10.0.
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 Com-
mittee at ICDDR,B had previously reviewed and approved
a general protocol for Nipah surveillance and response to
The outbreak occurred in two adjoining Districts of Man-
ikgonj and Rajbari over the same 6-day period in February
2008 (Fig. 2). We identified a total of 10 case-patients: 4 from
Table 1. Profiles of the Case-Patients in February
2008 Nipah Outbreaks, Manikgonj and Rajbari
CharacteristicsTotal, n=10; no. (%)
Mean (SD) in years
Median (range) in years
Altered mental status
Loss of consciousness
Difficulty in breathing
New onset of seizures
Onset of illness to death (n=9)
Mean (range) in days
Category of cases
NIPAH VIRUS OUTBREAK IN BANGLADESH, 2008 67
Manikgonj and 6 from Rajbari Districts. Nine of them died
(90%); one 12-year-old child from Rajbari survived. The me-
dian 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
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%) con-
firmed and six (60%) probable case-patients. Table 2 shows
the laboratory results of each case-patient.
Sequencing of the complete NiV nucleoprotein (N) ORF
amplified from these two isolated viruses from conven-
tional 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).
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 mem-
bers 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).
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
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
Table 2. Laboratory Results of Case-Patients in February 2008, Manikgonj and Rajbari Districts, Bangladesh
No Site Category
illness onset IgM IgG Isolation PCR
illness onset IgM IgG Isolation PCR
illness onset IgM IgG Isolation PCR
illness onset IgM IgG Isolation PCR
Neg Neg Neg
ND ND Neg
Neg Neg Neg
ND ND Neg
Neg Neg Neg
ND ND Pos
Neg Neg Neg
ND ND Pos
Neg Neg Neg
ND ND Neg
NA NA NA
NA NA NA
NA NA NA
NA 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
68RAHMAN ET AL.
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 pur-
chased from the neighborhood date palm sap collector with
two other neighborhood residents (brother-in-law and ne-
phew of that mother) on February 18; all five subsequently
developed illness. A salesman who resided nearly 5km 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.
Table 3. Nucleotide Differences Between Nipah Virus Isolated from Bangladesh (2004, 2008) and India (2007)
Nipah virus isolate IDNucleotide position
NIVBGD2004RAJBARI1 AY988601 serves as consensus sequence by which to compare the others.
‘‘.’’ indicates nucleotide identity with consensus sequence.
Table 4. Bivariate Analysis of Exposures for Nipah Virus Infection in February 2008, Manikgonj
and Rajbari Districts, Bangladesh
Case patients with
n=10; no. (%)
with this exposure,
n=40; no. (%)p-Value
Drank raw date palm sap
From local vendor
From any house
From any other sources
Collected from tree
Date sap harvesting
Date palm trees
Physical contact with living animals
Physical contact with sick animals
Physical contact with dead animals (chicken)
Ate sick animals
Ate dropped fruit
Local plum (Boroi)
Traveled outside subdistrict
Touched persons with fever and altered
mental status who died later
Been in the same room with persons with
fever and altered mental status who died later
10 (100) 10 (25)
6 (60)4 (10)0.656 2.20.3–19
2 (20)3 (8)0.258 2.90.2–30
Bolded type indicate significant results.
NIPAH VIRUS OUTBREAK IN BANGLADESH, 2008 69
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 100mL 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 associ-
ated 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–N, 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 multi-
variate 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.
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 Bangla-
desh (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, in-
cluding 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 infec-
tion. 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 con-
tacted 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 temper-
ature up to 22?C, is 18h (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 Ban-
gladesh, winter is the peak collection period of date palm
sap and this Nipah outbreak corresponds with the season-
ality of all previously reported Nipah outbreaks in Ban-
gladesh. 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
The date palm sap samples that were evaluated for the
presence of NiV were collected 24 days after the onset of ill-
ness 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 trans-
mission event suggests that date palm sap is only intermit-
tently contaminated, a pattern of contamination that is
consistent with the observed intermittent outbreaks in Ban-
The distance between the two areas (44km) was within the
50km 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 do-
mestic 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 col-
lected 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 sensi-
tized our study population regarding their response, but
70RAHMAN ET AL.
subjects andtheirproxiestorecalltheirexposurehistory more
elaborately, rather than encouraging them to hide those ex-
posures. 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 Gov-
ernment of Bangladesh. To prevent this illness, date palm sap
should not be drunk fresh unless effective steps have been
working with local date palm sap collectors to develop so-
cially 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, afford-
able 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 acceptableapproachestoproducesafedate
palm sap may provide an additional lifesaving prevention
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 com-
mitment 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.
No competing financial interests exist for any of the
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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Address correspondence to:
Muhammad Aziz Rahman
Discipline of Public Health
The University of Adelaide
Level-9, 10 Pulteney St.
72RAHMAN ET AL.