Articles

Association of acute toxic encephalopathy with litchi

consumption in an outbreak in Muzaffarpur, India,
2014: a case-control study
Aakash Shrivastava, Anil Kumar, Jerry D Thomas, Kayla F Laserson, Gyan Bhushan, Melissa D Carter, Mala Chhabra, Veena Mittal, Shashi Khare,
James J Sejvar, Mayank Dwivedi, Samantha L Isenberg, Rudolph Johnson, James L Pirkle, Jon D Sharer, Patricia L Hall, Rajesh Yadav,
Anoop Velayudhan, Mohan Papanna, Pankaj Singh, D Somashekar, Arghya Pradhan, Kapil Goel, Rajesh Pandey, Mohan Kumar, Satish Kumar,
Amit Chakrabarti, P Sivaperumal, A Ramesh Kumar, Joshua G Schier, Arthur Chang, Leigh Ann Graham, Thomas P Mathews, Darryl Johnson,
Liza Valentin, Kathleen L Caldwell, Jeffery M Jarrett, Leslie A Harden, Gary R Takeoka, Suxiang Tong, Krista Queen, Clinton Paden, Anne Whitney,
Dana L Haberling, Ram Singh, Ravi Shankar Singh, Kenneth C Earhart, A C Dhariwal, L S Chauhan, S Venkatesh, Padmini Srikantiah

Summary
Background Outbreaks of unexplained illness frequently remain under-investigated. In India, outbreaks of an acute Lancet Glob Health 2017
neurological illness with high mortality among children occur annually in Muzaffarpur, the country’s largest litchi Published Online
cultivation region. In 2014, we aimed to investigate the cause and risk factors for this illness. January 30, 2017
http://dx.doi.org/10.1016/
S2214-109X(17)30035-9
Methods In this hospital-based surveillance and nested age-matched case-control study, we did laboratory
See Online/Comment
investigations to assess potential infectious and non-infectious causes of this acute neurological illness. Cases were http://dx.doi.org/10.1016/
children aged 15 years or younger who were admitted to two hospitals in Muzaffarpur with new-onset seizures or S2214-109X(17)30046-3
altered sensorium. Age-matched controls were residents of Muzaffarpur who were admitted to the same two hospitals National Centre for Disease
for a non-neurologic illness within seven days of the date of admission of the case. Clinical specimens (blood, Control, India, Directorate
General of Health Services,
cerebrospinal fluid, and urine) and environmental specimens (litchis) were tested for evidence of infectious
Ministry of Health and Family
pathogens, pesticides, toxic metals, and other non-infectious causes, including presence of hypoglycin A or Welfare, Government of India,
methylenecyclopropylglycine (MCPG), naturally-occurring fruit-based toxins that cause hypoglycaemia and metabolic Delhi, India (A Shrivastava PhD,
derangement. Matched and unmatched (controlling for age) bivariate analyses were done and risk factors for illness A Kumar MD, M Chhabra MD,
V Mittal MD, S Khare MD,
were expressed as matched odds ratios and odds ratios (unmatched analyses).
R Singh PhD,
R Shankar Singh MD,
Findings Between May 26, and July 17, 2014, 390 patients meeting the case definition were admitted to the two referral L S Chauhan DPH,
hospitals in Muzaffarpur, of whom 122 (31%) died. On admission, 204 (62%) of 327 had blood glucose concentration S Venkatesh MD); National
Center for Environmental
of 70 mg/dL or less. 104 cases were compared with 104 age-matched hospital controls. Litchi consumption (matched
Health, US Centers for Disease
odds ratio [mOR] 9·6 [95% CI 3·6 – 24]) and absence of an evening meal (2·2 [1·2–4·3]) in the 24 h preceding illness Control and Prevention,
onset were associated with illness. The absence of an evening meal significantly modified the effect of eating litchis Atlanta, GA, USA
on illness (odds ratio [OR] 7·8 [95% CI 3·3–18·8], without evening meal; OR 3·6 [1·1–11·1] with an evening meal). (J D Thomas MD, M D Carter PhD,
R Johnson PhD, J L Pirkle MD,
Tests for infectious agents and pesticides were negative. Metabolites of hypoglycin A, MCPG, or both were detected in J G Schier MD, A Chang MD,
48 [66%] of 73 urine specimens from case-patients and none from 15 controls; 72 (90%) of 80 case-patient specimens L Valentin PhD, K L Caldwell PhD,
had abnormal plasma acylcarnitine profiles, consistent with severe disruption of fatty acid metabolism. In 36 litchi J M Jarrett MS); Global Disease
arils tested from Muzaffarpur, hypoglycin A concentrations ranged from 12·4 μg/g to 152·0 μg/g and MCPG ranged Detection Program, India, US
Centers for Disease Control and
from 44·9 μg/g to 220·0 μg/g. Prevention, Embassy of the
United States, Shanti Path,
Interpretation Our investigation suggests an outbreak of acute encephalopathy in Muzaffarpur associated with both Chanakyapuri, New Delhi, India
hypoglycin A and MCPG toxicity. To prevent illness and reduce mortality in the region, we recommended minimising (K F Laserson ScD,
M Dwivedi MD, R Yadav MBBS,
litchi consumption, ensuring receipt of an evening meal and implementing rapid glucose correction for suspected A Velayudhan MBBS,
illness. A comprehensive investigative approach in Muzaffarpur led to timely public health recommendations, M Papanna MD, K C Earhart MD,
underscoring the importance of using systematic methods in other unexplained illness outbreaks. P Srikantiah MD); Muzaffarpur
District Health Department,
Government of Bihar, Sadar
Funding US Centers for Disease Control and Prevention. Hospital, Muzaffarpur, Bihar,
India (G Bhushan MD); National
Copyright © The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND Center for Emerging and
license. Zoonotic Infectious Diseases,
US Centers for Disease Control
and Prevention, Atlanta, GA,
Introduction outbreaks begin in mid-May and peak in June, coinciding USA (J J Sejvar MD,
In India, seasonal outbreaks of an acute unexplained with the month-long litchi harvesting season. Children D L Haberling MSPH); Battelle at
the Centers for Disease Control
neurological illness have been reported since 1995 from from poor socioeconomic backgrounds in rural
and Prevention, Atlanta, GA,
Muzaffarpur, Bihar, the largest litchi (lychee) fruit Muzaffarpur comprise most of those affected. Illness is USA (S L Isenberg PhD,
cultivation region in the country.1 These recurring characterised by acute seizures and changed mental L A Graham PhD,

www.thelancet.com/lancetgh Published online January 30, 2017 http://dx.doi.org/10.1016/S2214-109X(17)30035-9 1

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T P Mathews PhD); Department

of Human Genetics, Emory Research in context
University, Decatur, GA, USA
(J D Sharer PhD, P L Hall PhD); Evidence before this study Added value of this study
India Epidemic Intelligence We searched PubMed between Jan 30, and April 30, 2013, This study, to the best of our knowledge, is the largest
Service, National Centre for before our 2013 field investigation, for any studies related to the investigation of the Muzaffarpur outbreak and the first
Disease Control, India,
acute unexplained neurological illness in Muzaffarpur using the comprehensive confirmation that this recurring outbreak illness
Directorate General of Health
Services, Ministry of Health search terms “Muzaffarpur,” AND (“encephalitis” OR is associated with litchi consumption and toxicity from both
and Family Welfare, “encephalopathy” OR “seizure.”) This identified two articles that hypoglycin A and MCPG. We confirm the presence of MCPG and
Government of India, Delhi, suggested potential causes for the outbreak illness varying from hypoglycin in litchis, and, for the first time, our data show the
India (R Yadav, A Velayudhan,
M Papanna, P Singh,
Japanese Encephalitis virus, another unknown virus, to heat metabolites of these toxins in human biological specimens, the
D Somashekar MD, stroke. Following the results of our 2013 investigation, which biological impact of these toxins on human metabolism, and
A Pradhan MBBS, K Goel MD, suggested hypoglycaemia might be an important factor in the modifying effect of the lack of an evening meal on the
R Pandey MBBS, M Kumar MBBS, illness, and raised the possibility of a toxic origin, we repeated a impact of these toxins.
S Kumar MD); National
Institute of Occupational
PubMed search in December, 2013, using terms (“ackee
Implications of all the available evidence
Health, Indian Council of hypoglycin” OR “ackee encephalopathy” OR “glycine analog
Based on the results of our investigation, public health and
Medical Research, Ministry AND litchi” OR “litchi encephalopathy” or “litchi
of Health and Family Welfare, clinical recommendations targeted at preventing illness and
methylenecyclopropylglycine” (MCPG), OR “Jamaican vomiting
Government of India, Meghani reducing morbidity and mortality from the Muzaffarpur
Nagar, Ahmedabad, Gujarat,
sickness”) for studies describing an association between litchis,
outbreak illness were provided to state and national health
India (A Chakrabarti MD, ackee fruit, hypoglycin, MCPG and acute neurologic illness
authorities. This included recommendations to minimise litchi
P Sivaperumal PhD, published between Jan 1, 1954, and Dec 1, 2013. We found
A R Kumar PhD); Oak Ridge consumption among young children in the affected area, to
61 studies; 11 described cases or outbreaks of ackee fruit
Institute for Science and ensure that children receive an evening meal throughout the
Education Fellow at the Centers
poisoning which implicated hypoglycin toxicity; an additional
outbreak period, and to rapidly assess and correct
for Disease Control and 11 studies described the pathophysiology of hypoglycin A in
hypoglycaemia in any child suspected of having the outbreak
Prevention, Atlanta, GA, USA animal models, and five described methods to characterise
(D Johnson PhD); Western illness. Evaluation of other potential factors, including missed
hypoglycin A in ackee fruits. An ecological study from 2012 from
Regional Research Center, evening meal, poor nutritional status, and as yet unidentified
US Department of Agriculture, Vietnam indicated an association between litchi plantation
genetic differences, may provide further insights into additional
Albany, CA, USA surface area and acute encephalitis incidence. A study from
risk factors for this outbreak illness. Application of a similar
(L A Harden MS, 1962 described the isolation of MCPG in litchi seeds, and
G R Takeoka PhD); National comprehensive and systematic approach to the evaluation of
two studies from 1989 and 1991 described the hypoglycaemic
Center for Immunizations and both infectious and non-infectious aetiologies of unexplained
Respiratory Diseases, effect of MCPG in animal studies. No studies implicated a direct
illness outbreaks in other parts of the world has the potential to
US Centers for Disease Control epidemiological association between litchi consumption in
and Prevention, Atlanta, GA,
contribute toward identifying interventions that can reduce
affected individuals and encephalopathy. No studies showed
USA (S Tong PhD, K Queen PhD, morbidity and mortality.
hypoglycin or MCPG or their metabolites in affected individuals.
C Paden PhD, A Whitney PhD);
Center for Global Health, US
Centers for Disease Control and
Prevention, Atlanta, GA, USA status, frequently with onset in the early morning,2 and is admission, which was also associated with increased
(K F Laserson, K C Earhart, associated with high mortality. A wide spectrum of causes mortality. These findings focused our attention on the
P Srikantiah); and National
Vector Borne Disease Control
has been proposed for this illness, including infectious possibility that children in Muzaffarpur were exposed to
Programme, Directorate encephalitis, exposure to pesticides, and a potential an environmental toxin, which resulted in low blood
General of Health Services, association with litchi fruit consumption.3–6 Despite glucose and, subsequently, seizures and encephalopathy.
Ministry of Health and Family numerous investigations, neither a cause nor risk factors Published reports of a toxic hypoglycaemic syndrome
Welfare, Government of India,
Nirman Bhavan, New Delhi,
for illness have been confirmed among affected in the West Indies8–10 that was due to the effects of
India (A C Dhariwal MD) individuals. hypoglycin A, a toxin found in the ackee, which is a fruit
Correspondence to: In 2013, the National Centre for Disease Control, India in the same botanical family as litchi, raised the prospect
Dr Padmini Srikantiah, Global (NCDC) and the US Centers for Disease Control and of a litchi-associated toxin. Of specific interest was the
Disease Detection Program, Prevention (US CDC) initiated an investigation, focusing potential role of methylenecyclopropylglycine (MCPG), a
India, US Centers for Disease
Control and Prevention, Embassy
on characterising the clinical and epidemiological features homologue of hypoglycin A, and a substance naturally
of the United States, Shanti Path, of illness, and assessing potential infectious causes. The found in the litchi seed and fruit known to cause
Chanakyapuri, New Delhi, India laboratory investigation found no evidence of a known hypoglycaemia in animal studies by inhibiting β-oxidation
[email protected] infectious cause, and clinical data indicated that the illness of fatty acids and gluconeogenesis.3,6,11,12 We aimed to
was consistent with a non-inflammatory encephalopathy.7 investigate the cause and risk factors for this illness.
These results led to consideration of various non-
infectious causes, including pesticides or herbicides used Methods
to spray orchards and agricultural fields, insecticides used Study design
in vector-borne disease control efforts, heavy metals, or In 2014, NCDC and US CDC investigated this syndrome,
exposure to unusual medications. Notably, a common using hospital-based clinical surveillance, an epidemio-
laboratory finding was low blood glucose (<70 mg/dL) on logical case-control study, and comprehensive and novel

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laboratory testing methods on human biological and interest, including litchi orchards and litchis, we were
environmental specimens to determine risk factors concerned about the possibility of overmatching. To
associated with this illness, assess the aetiological role of prevent this, both community (adjacent village) and
naturally occurring toxins such as MCPG and hypoglycin, hospital controls (any other village) were selected from
and exclude the role of novel infectious pathogens, villages other than the case-patients; the community
selected pesticides, and toxic elements. controls were subsequently assessed to still be over-
matched and were thus dropped from the analysis. A
Hospital-based clinical surveillance hospital control was defined as a resident of Muzaffarpur
Surveillance was done at the Shri Krishna Medical College district who was admitted to one of the surveillance
Hospital (SKMCH) and the Krishnadevi Deviprasad hospitals for a non-neurological illness within 7 days of
Kejriwal Maternity Hospital (KDKMH), the chief referral the date of admission of the case. Children who had a
medical centers in Muzaffarpur district, India. history of altered mental status or seizures in the previous
A case was defined as new-onset seizures or altered 3 months were excluded as controls. For case-patients
sensorium in the previous seven days in a child aged younger than 5 years, hospital controls were age-matched
15 years or younger admitted to either SKMCH or to within 6 months of age; for case-patients who were
KDKMH. Patients admitted for febrile seizures, defined 5 years or older, controls were age-matched to within
as a seizure in a child 6 months to 6 years whose only 12 months of age. Informed consent was obtained from
finding is fever, and a single generalised convulsion of parents or guardians.
less than 15 min duration who recovers consciousness Cases and controls were asked about consumption of
within 60 min of the seizure13 were excluded. Ill children food items, food washing, water sources, and other
who met the case definition and were admitted at either exposures, including time spent in agricultural fields.
of the two referral hospitals in Muzaffarpur were Standardised data for household characteristics,
prospectively enrolled. Demographic and clinical data ownership of household assets or goods, and land were
were collected with standardised questionnaires. collected to calculate a socioeconomic index (SEI)
According to district level clinical guidelines, a according to the methods of the National Family Health
patient’s blood glucose was assessed at presentation, Survey, a large-scale, multiround survey undertaken
ideally before administration of any treatment; treating throughout India by the Ministry of Health and Family
clinicians provided intravenous dextrose therapy to all Welfare.14,15 Data for both case and controls were
patients suspected to have the outbreak illness. Lumbar systematically collected using standardised
puncture was done according to the clinician’s decision; questionnaires. Bodyweight (kg) and body height or
cytological (white blood cell [WBC] count) and length (cm) were measured for each enrolled case and
biochemical (protein and glucose) examination were control. A child was defined as wasted if the Z score was
done on collected CSF specimens. Blood and urine more than 2 SD below WHO Child Growth Standards16
specimens were collected on all enrolled patients at the of calculated body-mass index (BMI; children ≥5 years of
time of admission. Detailed neurological examination age) or weight for height (children <5 years of age), and
was done within 12 h of admission on a subset of case- stunted if the Z score was more than 2 SD
patients. Brain MRI (including fluid attenuation below the same standards of calculated BMI (children
inversion recovery [FLAIR] sequence) and EEG ≥5 years of age) or height for age (children <5 years of
diagnostic testing, not normally available at the treating age). Additionally, urine and blood specimens were
hospitals, were done when possible. collected from each enrolled control. Each case-patient
and control household was visited to collect data for
Case-control study observed exposures.
Every alternate surveillance case-patient who survived at
least 6 h beyond the time of admission was prospectively Environmental specimen collection
enrolled in an age-matched case control study if he or she Between May 19, and June 13, 2014, litchi fruit samples
was a resident of Muzaffarpur district. We calculated a were collected from orchards in the five blocks of
sample size of 100 cases and 200 controls, assuming Muzaffarpur district with the highest reported number of
80% power, 50% exposure of the key risk factors among cases in 2013 and 2014. In each block, six or more fruits
controls, and ratio of controls to cases of 2:1. Due to a were collected in each of the following categories: unripe,
rapid increase in cases and restricted human resources, ripe plucked from tree, and ripe fallen on the ground.
enrolment was modified on June 16, 2014, to every fourth Each fruit was stored at –20°C within 3 h of collection and
eligible case-patient to attain the calculated sample size subsequently transferred to –70°C until analysis.
and have continuous enrolment throughout the outbreak
period. For each case, we initially enrolled one community Laboratory testing
control and one hospital control within 7 days of case CSF and serum specimens from case-patients were tested
enrolment. In view of the overall homogeneity of rural at NCDC using PCR for viruses, including Japanese
Muzaffarpur and the ubiquitous nature of the variables of encephalitis virus, West Nile virus, and enteroviruses.7 A

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subset of case-patient CSF and serum specimens collected was obtained in the local language (Hindi) from the
in both the 2013 and 2014 investigations was submitted for parent or guardian of each child enrolled. While
assessment of additional infectious agents, including laboratory testing on collected case-patient CSF
potential novel pathogens, to the US CDC Pathogen specimens was done as part of the investigation, the
See Online for appendix Discovery Laboratory (Atlanta, GA, USA;17,18 appendix p 4). decision of whether or not to collect CSF was solely made
Blood and urine specimens of cases from 2014 were examined by the treating physician based on his or her clinical
at the US CDC for metabolites of pesticides and toxic judgment. Participants and their parents or guardians
elements using established mass spectrometry methods.19–23 were informed that some laboratory test results would
At the National Institute of Occupational Health, India only be available months later, and, although not of
(NIOH), red blood cell acetylcholinesterase and plasma specific immediate benefit to the participating child,
butyryl cholinesterase activity were measured24 (appendix could help health officials to understand the cause of the
p 5), and litchi fruit samples were analysed for pesticide outbreak, and thus benefit the community. In 2015, when
residues using the Quick Easy Cheap Effective Rugged and final laboratory results were available from NCDC and
Safe method25 (appendix p 8). A novel assay was developed US CDC, these results were communicated to district
at US CDC to analyse case and control specimens health officials and treating clinicians who conveyed
from 2013 and 2014 for metabolites of hypoglycin A them to participating families.
and MCPG using liquid chromatography-tandem mass
spectrometry.26 Plasma acylcarnitine and quantitative and Statistical analysis
qualitative urine organic acid profiles were assessed at the Data were entered in Epi-Info version 7.0 (CDC, Atlanta,
Emory Genetics Laboratory (Atlanta, GA, USA) using GA, USA) and analysed with Stata version 13.0 (Stata,
established mass spectrometry methods27–31 to identify College Station, Texas, USA) and SAS/STAT software
evidence of derangement in fatty acid metabolism, which version 9.3 (SAS Institute Inc, Cary, NC, USA). Matched
was postulated to occur in the case of MCPG or hypoglycin bivariate analyses as well as unmatched bivariate analyses
A toxicity as a result of impaired β-oxidation11,12,32 (appendix controlling for age were done; risk factors for illness
p 9). Laboratory scientists were blinded to case or control expressed as matched odds ratios (mOR; matched
designation of the specimens under assessment. In a analysis) and odds ratios (OR; unmatched analysis) with
collaboration between the US Department of Agriculture 95% CI. Potential interactions between exposures were
(USDA) and the US CDC, a quantitative assay was designed examined in stratified analyses, controlled for age. A
to assess MCPG and hypoglycin A content in soapberry p value less than 0·05 was considered significant.
arils33 (appendix p 10).
Role of the funding source
Ethical approval The funder had no role in study design; in the
Ethical approval for this investigation and case-control collection, analysis, or interpretation of data; in the
study was obtained from the institutional review boards writing of the report; or in the decision to submit the
of NCDC and the US CDC. Written informed consent paper for publication. The corresponding author had
full access to all the data in the study and all authors
had final responsibility for the decision to submit for
n/N (%)
publication.
Men 213/390 (55%)
Age (years)
Results
<1 8/390 (2%) Between May 26, and July 17, 2014, 390 patients meeting
1–5 280/390 (72%) the case definition were admitted to the two referral
6–10 98/390 (25%) hospitals in Muzaffarpur. Among these, 213 (55%) were
11–15 4/390 (1%) boys, median age was 4 years (range 6 months–14 years),
Mortality 122/386 (32%) and 280 (72%) were aged 1–5 years (table 1). Among case-
Generalised seizure at presentation 326/348 (94%) patients with recorded measurements, 11 (16%) of 68 were
Altered mental status at presentation 345/362 (95%) classified as wasted and 46 (65%) of 71 were classified as
Afebrile (≤37·5°C) on admission 219/357 (61%) stunted. Most patients (273; 70%) were from Muzaffarpur
Illness onset between 0300–0800 h 224/342 (66%) district; cases were reported from all 16 blocks of
Blood glucose ≤3·89 mmol/L on admission 204/327 (62%) Muzaffarpur district. Clustering of cases was not observed;
CSF cytology <0·5 x 106 WBC/L 52/62 (84%) each affected child seemed to be an isolated case in a village
CSF protein <450 mg/L 58/62 (94%) (approximate population per village 2500). The outbreak
CSF glucose >2·50 mmol/L 49/62 (79%) peaked in mid-June, with 147 cases reported during
Brain MRI with no focal lesions 16/16 (100%) June 8–14, 2014, and declined substantially after
EEG consistent with generalised encephalopathy 22/30 (73%) June 21, 2014 (figure).
Caregivers reported that affected children were
Table 1: Characteristics of case patients, Muzaffarpur, May–June, 2014
previously well and 366 (94%) had sudden onset of

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symptoms less than 24 h before admission. Further, 30 Death

224 (66%) of 342 patients with recorded data reported Case
illness onset between 0300 h and 0800 h. Of patients 25
with recorded data, 326 (94%) of 348 reported one or
more seizures and 345 (95%) of 362 reported altered 20

Number of cases
mental status before admission; 301 (87%) of 347 patients
were unconscious on presentation. Seizure semiology 15
was characterised by intermittent generalised tonic or
tonic-clonic seizures; duration and frequency of the 10
seizures varied (appendix p 10). Several patients had
convulsive or non-convulsive status epilepticus. 5
Vomiting was reported in 59 (18%) of 337 patients with
recorded data. Of 357 patients with recorded admission
0
measurements, the median temperature was 37·2ºC 26 27 28 29 30 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
May June July
(99ºF; range 35·6–40·6), and 219 (61%) were afebrile Date of admission
(≤37·5ºC [≤99·5º ]). Among 386 patients with recorded
Figure: Acute neurological illness by date of hospital admission in Muzaffarpur, May 29–July 17, 2014
data, 122 died (case fatality rate 32%).
On detailed clinical assessment of 52 patients, 48 (92%)
showed no focal neurological deficits. Brain MRI of
Cases (N=104) Controls (N=104) mOR (95% CI)
16 patients showed no focal lesions, signal abnormalities,
or changes suggestive of inflammation; eight patients Ate litchi* 67/103 (65%) 23/102 (23%) 9·6 (3·8–24·1)
(50%) showed mild to moderate cerebral oedema. Visited fruit orchard* 52/100 (52%) 18/98 (32%) 6·0 (2·7–13·4)
Clinical severity did not noticeably differ between Parent visited fruit orchard* 29/95 (31%) 16/99 (16%) 2·3 (1·1–4·8)
participants with and without cerebral oedema. EEG in Absence of evening meal* 76/98 (78%) 51/88 (58%) 2·2 (1·2– 4·3)
30 cases showed findings consistent with generalised Socioeconomic index below poverty line 57/104(55%) 49/104 (47%) 1·4 (0·8–2·4)
encephalopathy in 22 (73%); seven showed epileptiform Routinely wash vegetables and fruits 32/99 (32%) 58/83 (70%) 0·13 (0·05–0·4)
discharges. Of 62 patients with CSF collected for analysis,
mOR=matched odds ratio.*In 24 h before symptom onset.
52 (84%) had normal WBC counts (<0·5 x 10⁶ cells per L),
58 (94%) had normal protein (<450 mg/L), and 49 (79%) Table 2: Exposures associated with illness in matched bivariate analysis of case control study in Muzaffarpur,
had normal glucose (>2·50 mmol/L) concentrations. Of June–July, 2014
327 patients with blood glucose measurement on
admission, the median blood glucose level was [non-litchi] meal before 1900 h; mOR 2·2 [95% CI
2·66 mmol/L (range 0·44–23·98), and 100 (31%) patients 1·2–4·3]) in the 24 h preceding illness onset (table 2),
had glucose concentration of 1·67 mmol/L or less, and were similar to what was noted in unmatched
171 (52%) patients had glucose concentration of bivariate analyses controlled for age (appendix p 11).
2·78 mmol/L or less, and 204 (62%) patients had glucose Calculated socioeconomic index did not differ between
concentrations of 3·89 mmol/L or less. Of 349 patients cases and controls (mOR 1·4 [95% CI 0·8–2·4]);
with available information, 239 (69%) had a record of routinely washing vegetables and fruits (mOR 0·1
receiving dextrose therapy during hospital stay; of these, [0·05–0·4) could be protective. Among those who
173 (73%) survived. consumed litchis, cases were more likely to eat unripe
Of 331 patients with recorded data, 149 (45%) were litchis (mOR 7·9 [95% CI 1·1–347·0), eat rotten litchis
referred from another health-care facility, such as a (7·4 [1·5–69·8]), report eating litchis from the ground
primary health centre or private clinic; the remainder versus from the tree (22 cases vs no controls), and
presented directly to the referral hospitals. In a report eating partially eaten litchis (17 cases vs no
multivariable model controlling for hypoglycaemia, controls).
presence of fever on admission, and receipt of dextrose Other factors, including biting, eating, or chewing the
therapy during hospital stay, patients referred to the litchi seed and peeling or eating the litchi peel were not
hospital from another health facility were twice as likely associated with illness (data not shown). Similarly, no
to die as those who came directly to the referral hospital association was noted between illness and consumption
(OR 2·3 [95% CI 1·2–4·1]). of raw vegetables or medications, drinking water source,
Between June 1, and July 10, 2014, 104 cases and or exposure to insecticides or chemicals sprayed in and
104 age-matched hospital controls were enrolled. around the house or nearby fields or orchards (data not
Exposures that were significantly associated with illness shown). For children younger than 5 years, mean Z scores
on matched bivariate analysis included litchi for height for age (–2·85 [cases] vs –2·18 [controls],
consumption (matched odds ratio [mOR] 9·6 [95% CI p=0·12) and weight for height (0·00 [cases] vs
3·8–24·1]), visiting a fruit orchard (6·0 [2·7–13·4]), and –1·00 [controls], p=0·08) did not significantly differ
absence of an evening meal (defined as eating the last between cases and controls. Among children older than

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metabolites. Creatinine-corrected concentrations of

Cases Controls
MCPF-Gly and MCPA-Gly were determined for each
Abnormal acylcarnitine profile 72/80 (90%) NA sample as μg of metabolite per g of creatinine (μg/g-cr).
Abnormal urine organic acid profile 67/75 (89%) 0/15 (0%) For MCPF-Gly, the 33 positive samples ranged from
Urinary metabolite for hypoglycin A 47/73 (64%) 0/15 (0%) 0·289 to 6·80 × 10³ μg/g-cr, with a median of
Urinary metabolite for MCPG 33/73 (45%) 0/15 (0%) 1·22 × 10³ μg/g-cr. For MCPA-Gly, the 47 positive samples
Data are n/N (%). NA=not available. MCPG=methylenecyclopropylglycine. ranged from 0·0402 to 1·89 × 10⁴ μg/g-cr, with a median
of 2·63 × 103 μg/g-cr (table 3). On assessment, 67 (89%) of
Table 3: Analysis of acylcarnitine, organic urinary acids, and metabolites 75 specimens showed abnormal urinary organic acid
of hypoglycin A and MCPG in cases and controls in Muzaffarpur, 2013–14
profiles and 72 (90%) of 80 specimens had abnormal
plasma acylcarnitine profiles, consistent with severe
MCPG (μg/g dry weight) Hypoglycin A (μg/g dry weight) disruption of fatty acid metabolism. None of the 15 control
1 specimens tested showed abnormal urinary organic acid
Ripe 66·4 74·1 profiles nor tested positive for reportable concentrations
Unripe 220·0 152·0 of either the hypoglycin A or MCPG metabolite.
2 Of 36 litchi arils analysed from Muzaffarpur, observed
Ripe 68·0 50·5 concentrations ranged from 12·4 μg/g to 152·0 μg/g
Unripe 112·0 136·0 hypoglycin A and 44·9 μg/g to 220·0 μg/g MCPG
3 (table 4). Within each batch tested, the unripe fruit
Ripe 44·9 12·4 contained higher concentrations of both MCPG and
Unripe 82·1 18·5
hypoglycin A than did the ripe fruit.

MCPG=methylenecyclopropylglycine. Both the ripe and unripe groups contained Discussion

6 homogenates.
Although an association with MCPG has been previously
Table 4: Analysis of hypoglycin A and MCPG in litchi fruit arils in proposed,2,3,34 and MCPG has been detected in the seed
Muzaffarpur, 2014 and aril of the litchi,6,35 this is the first confirmation that
this recurring outbreak in Muzaffarpur is associated with
5 years, the mean Z score for calculated BMI did not litchi consumption and both hypoglycin A and MCPG
differ significantly between cases and controls toxicity. This conclusion is supported by clinical findings
(–0·81 [cases] vs –1·90 [controls], p=0·08). consistent with an acute toxic encephalopathy, significant
On stratified analysis controlled for age, the absence of epidemiological association between litchi consumption
an evening meal in the previous 24 h significantly and illness, laboratory results that show, for the first time
modified the relation between litchi consumption and to our knowledge, the presence of hypoglycin A and
illness (OR 7·8 [95% CI 3·3–18·8], without evening MCPG metabolites, and evidence of resultant metabolic
meal; OR 3·6 [95% CI 1·1–11·1] with evening meal). derangement in the biological specimens of cases but
At NCDC, laboratory diagnostic testing of 17 CSF not controls, and the confirmation of these toxins in
specimens for Japanese encephalitis virus and West litchi fruits. The absence of clinical, epidemiological, or
Nile virus virus by PCR, and an additional 12 CSF laboratory findings to support infectious pathogen,
specimens with an 11-virus multiplex PCR platform pesticide, and heavy metal related causes of illness
assay were negative. Pan-viral family or genus PCRs and suggest the observed protective association of routinely
sequencing of 40 CSF and 40 serum samples at US CDC washing fruit or vegetables was not directly related to a
showed one CSF sample and one serum sample (from toxin or infectious agent.
two different patients) were positive for Adenovirus 41. Findings of organic acid and acylcarnitine analysis
A separate CSF specimen tested positive for a divergent showed evidence of disruption of several dehydrogenase
rhabdovirus. All other samples were negative for all enzymes involved in fatty acid oxidation, similar to profiles
assays tested. observed in glutaric acidemia type II, an inherited
No pattern of excessive pesticide or metal exposures metabolic disorder with a panethnic prevalence that is less
was identified in 80 case-patient specimens examined at than 1:100 000.36 However, the temporal and spatial
US CDC. No abnormality in acetylcholinesterase or concentration of case-patients observed in this outbreak is
butyrylcholinesterase activity levels was detected in the inconsistent with this specific genetic cause. Furthermore,
specimens of 27 patients examined at NIOH. Additionally, similar abnormal urinary organic acid profiles (increased
no pesticide residue was detected in 14 litchi samples ethylmalonic acid, glutaric acid, and adipic acid) have been
assessed at NIOH. reported in ackee fruit encephalopathy outbreaks,10,37
Among 73 case-patient urine specimens assessed, further supporting that the changes observed in patients in
47 (64%) contained metabolites of hypoglycin A Muzaffarpur are a result of disrupted fatty acid metabolism
(MCPA-Gly), 33 (45%) contained metabolites of MCPG due to hypoglycin A and MCPG toxicity. The acidosis
(MCPF-Gly), and 32 (44%) specimens contained both resulting from accumulation of certain fatty acids might

6 www.thelancet.com/lancetgh Published online January 30, 2017 http://dx.doi.org/10.1016/S2214-109X(17)30035-9

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have further contributed to clinical encephalopathy, which metabolic derangements and clinical manifestations);
could explain symptoms and signs observed even among 6) coherence between the laboratory and epidemiological
patients without documented hypoglycaemia, an event findings; and 7) analogy (similar reports and findings in
that has also been reported in ackee fruit encephalopathy.38 outbreaks of toxic encephalopathy due to ackee, a fruit in
Limitations in the ability to provide aggressive critical care, the same botanical family as litchi). Based on these
including closer respiratory monitoring and mechanical observations, we conclude that our findings reflect a
ventilation, probably contributed to mortality among plausible, but not necessarily sufficient, causal pathway
affected children, despite the administration of dextrose between litchi consumption and illness.
supplementation. Our findings support the need to Within India, an outbreak of a similar acute neurological
strengthen clinical intensive care capacity at the treating illness with hypoglycaemia and seizures was reported in
hospitals in Muzaffarpur. June, 2014, among young children in Malda, a litchi
Our analyses indicate that the absence of an evening cultivation district in West Bengal.43 In southeast Asia,
meal modified the association between litchi consumption outbreaks of similar acute neurological illnesses have also
and illness. Parents in affected villages report that during been reported from litchi-growing areas of Bangladesh
May and June, young children frequently spend their day and Vietnam.44,45 These outbreaks have not been similarly
eating litchis in the surrounding orchards; many return comprehensively investigated. The investigation in
home in the evening uninterested in eating a meal. Bangladesh focused on the possibility that pesticides
Skipping an evening meal is likely to result in night-time used seasonally in litchi orchards might be involved, but
hypoglycaemia, particularly in young children who have no specific pesticide was implicated. The investigation in
limited hepatic glycogen reserves, which would normally Vietnam focused on possible infectious agents that might
trigger β-oxidation of fatty acids for energy production and be present seasonally near litchi fruit plantations, but
gluconeogenesis.39,40 However, in the setting of hypoglycin found none to explain the outbreak. Our investigations
A/MCPG toxicity, fatty acid metabolism is disrupted and also thoroughly explored the possibilities of pesticide and
glucose synthesis is severely impaired,11,12,41 which can lead heavy metal related toxicity but found no clinical,
to the characteristic acute hypoglycaemia and encephalo- epidemiological, or laboratory evidence to support this.
pathy of the outbreak illness. The association between Detailed assessments of infectious causes, including for
illness and the absence of an evening meal could explain viral pathogens known to cause encephalitis in the region
the early morning onset of symptoms noted in most as well as for potential novel infectious agents, were also
patients, and supports recommendations to ensure that consistently negative. The findings of our investigations
children receive a night-time meal throughout the might help to shed light on the cause of illness in the
outbreak period. The important interaction between litchi Bangladesh and Vietnam outbreaks.
consumption and the absence of an evening meal also At a broader level, the Muzaffarpur outbreak is illustrative
contributes toward an understanding of why only some of unexplained public health threats in resource-
children in Muzaffarpur develop this acute encephalopathy. constrained settings, whether localised or regional, that are
Although litchi fruits are ubiquitous in the orchards frequently under-investigated. The application of a
surrounding the villages in rural Muzaffarpur, typically comprehensive multisectoral investigation in Muzaffarpur,
only one child in an entire village develops this acute with the combined inputs of clinicians, epidemiologists,
illness. The synergistic combination of litchi consumption, laboratory scientists, environmental specialists, and
a missed evening meal, and other potential factors such as medical toxicologists enabled the methodical exclusion of
poor nutritional status, eating a greater number of litchis, infectious pathogens, the consideration of potential
and as yet unidentified genetic differences might be environmental causes that had not previously been
needed to produce this illness. systematically assessed, and the comprehensive testing of
Although our findings show an association between both environmental and human specimens to investigate
hypoglycin A/MCPG toxicity, litchi consumption, and and confirm a postulated association between litchi fruits,
this outbreak illness, causality is considerably more hypoglycin A/MCPG, and illness that led to timely public
difficult to establish. Assessment of our results using the health recommendations to prevent illness and reduce
Bradford Hill criteria for causation42 showed that seven of mortality.46 Using similar systematic investigation
nine criteria are met: 1) strength of association (large methods, both in other countries affected by similar
ORs for consumption of litchi, modified by presence or outbreaks as well as in other settings of unexplained illness
absence of evening meal); 2) consistency (clinical has major potential to contribute toward improving public
findings shown in both 2013 and 2014, and MCPG health response.
detected in litchi fruit previously); 3) specificity (specific Quantitative evaluation of a small number of litchi arils
population, primarily young children, at a specific (edible fruit) collected in Muzaffarpur indicated
location, Muzaffarpur, affected, and no clear evidence for approximately twice the level of detected hypoglycin A, as
any other cause; 4) temporality (illness follows the litchi well as MCPG in unripe versus ripe fruits. This finding
harvest season); 5) plausibility (biological mechanism for is in contrast with what is seen in ackee fruit, where the
MCPG/hypoglycin A toxicity leading to the observed concentration of hypoglycin A in unripe fruits is more

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than 20 times higher than that observed in ripe fruits.47,48 interpreted testing for hypoglycin A and MCPG in litchi fruit samples. ST,
A larger quantitative evaluation of hypoglycin A and KQ, CP, and AW did and interpreted tests for novel infectious pathogens.
PS, AS, KL, AK analysed the clinical and epidemiological data. JJS, JGS,
MCPG concentrations in different cultivars as well as AC, and SV supported analysis and interpretation. JJS and DH did the
several stages of maturation is needed to better evaluate nutritional analyses. PS wrote the first draft of the report. PS, AS, KL, JJS,
this question. If substantial differences in the JDT, JLP, MDC, SLI, ST, GRT, LAH, and SV wrote the report. All authors
concentrations of these compounds are consistently discussed the results and contributed to revision of the final manuscript.
detected in different stages of litchis, public health Declaration of interests
prevention recommendations regarding litchi fruit We declare no competing interests.
consumption can be further refined. Acknowledgments
This study was subject to two major limitations. First, This study was supported by a CDC Research Project Cooperative
Agreement Grant numbers GH-10-002 U2G GH000066-01-05.
determination of whether litchi fruit had been consumed The findings and conclusions in this report are those of the authors and
before symptom onset relied upon reported information do not necessarily represent the official position of the US Centers for
from the parent or caregiver of the child, who might not Disease Control and Prevention. We thank the clinicians and
have been with the child during consumption. However, administrations of the Shri Krishna Medical College Hospital and the
Krishnadevi Deviprasad Kejriwal Maternity Hospital for their
both cases and controls were ill and in hospital and collaboration and support of this investigation, and their dedicated care of
queried about exposures before admission to hospital; affected patients. We are particularly grateful for the support of the
we, therefore, expect that both groups would have been pediatricians. We also thank the JE/AES Program of the National Vector
equally likely to report exposures such as food Borne Disease Control Program and the office of the Directorate General
of Health Services for their support. We are deeply indebted to the
consumption, thereby minimising the potential for patients and their families for their participation and cooperation.
differential misclassification. Additionally, the absence of
References
difference in socioeconomic status, as well as the overall 1 Ministry of Agriculture Government of India. Statewise area,
homogeneity observed throughout the 16 blocks of production, and productivity of Litchi: Indian Horticulture Database
2014. Gurgaon: National Horticulture Board, 2015.
Muzaffarpur district, suggests that both groups would be
2 Shah A, John TJ. Recurrent outbreaks of hypoglycaemic
presented with equal opportunity for exposure to encephalopathy in Muzaffarpur, Bihar. Curr Sci 2014; 107: 570–71.
variables of interest. Second, although our protocol 3 John TJ, Das M. Acute Encephalitis Syndrome in children in
required control interview and specimen collection Muzaffarpur: hypothesis of toxic origin. Curr Sci 2014 106: 1184–85.
4 Sahni GS. Recurring epidemics of acute encephalopathy in children
within a 7 day window of the matched case, this was not in Muzaffarpur, Bihar. Indian Pediatr 2012; 49: 502–03.
always possible and some delays occurred. 5 Samuel PP, Muniaraj M, Thenmozhi V, Tyagi BK.
In conclusion, to the best of our knowledge, this is the Entomo-virological study of a suspected Japanese encephalitis
first comprehensive confirmation that this recurring outbreak in Muzaffarpur district, Bihar, India. Indian J Med Res
2013; 137: 991–92.
outbreak of acute encephalopathy is associated with both 6 Das M, Asthana S, Singh SP, Dixit S, Tripathi A, John TJ. Litchi
hypoglycin A and MCPG toxicity from litchi fruit contains methylene cyclopropyl-glycine. Curr Sci 2015;
consumption. This illness is also associated with absence 109: 2195–97.
7 Shrivastava A, Srikantiah P, Kumar A, et al. Outbreaks of
of an evening meal. To prevent illness and save lives in unexplained neurologic illness - Muzaffarpur, India, 2013-2014.
Muzaffarpur, we recommended46 minimising litchi MMWR Morb Mortal Wkly Rep 2015; 64: 49–53.
consumption among young children, ensuring children 8 Centers for Disease Control. Toxic hypoglycemic syndrome—
Jamaica, 1989–1991. MMWR Morb Mortal Wkly Rep 1992; 41: 53–55.
in the area receive an evening meal throughout the
9 Gaillard Y, Carlier J, Berscht M, et al. Fatal intoxication due to ackee
outbreak season, and implementing rapid glucose (Blighia sapida) in Suriname and French Guyana. GC-MS detection
correction for children with suspected illness. Application and quantification of hypoglycin-A. Forensic Sci Int 2011; 206: e103–07.
of a similar comprehensive and systematic approach to 10 Joskow R, Belson M, Vesper H, Backer L, Rubin C. Ackee fruit
poisoning: an outbreak investigation in Haiti 2000-2001, and review
the assessment of both infectious and non-infectious of the literature. Clin Toxicol (Phila) 2006; 44: 267–73.
causes of unexplained illness outbreaks in other parts of 11 Melde K, Buettner H, Boschert W, Wolf HP, Ghisla S. Mechanism
the world can contribute greatly toward identifying of hypoglycaemic action of methylenecyclopropylglycine. Biochem J
1989; 259: 921–24.
interventions that can reduce morbidity and mortality.
12 Melde K, Jackson S, Bartlett K, Sherratt HS, Ghisla S.
Contributors Metabolic consequences of methylenecyclopropylglycine poisoning
AS, PS, KL, and JJS conceived the study design. AK, JT, JS, AC, LSC, and in rats. Biochem J 1991; 274: 395–400.
KE contributed to the study design. AS and PS led the field investigation. 13 Patterson JL, Carapetian SA, Hageman JR, Kelley KR.
KL, JJS, AK, GB, MD, RY, AV, MP, PS, DS, AP, KG, RP, MK, SK, RS, and Febrile seizures. Pediatr Ann 2013; 42: 249–54.
RSS facilitated and did the field epidemiological investigation. JJS did the 14 Adhikari T, Singh P. Development of Socio-Economic Indices for
neurological assessments. JDT, MDC, SLI, RCJ, JLP developed, did, and Identifcation of Poor. J Emp Res Social Sci 2007; 2: 1–12.
interpreted assays to evaluate and quantify the presence of hypoglycin A 15 Indian Institute of Population Studies. National Family Health Survey,
and MCPG metabolites as well as pesticide metabolites in human India. 2009. http://rchiips.org/nfhs/ (accessed March 15, 2016).
biological specimens, and assays to detect hypoglycin A in fruit samples. 16 WHO and UNICEF. WHO Child Growth Standards and the
JDS and PLH did and interpreted assays to evaluate the profiles of urinary Identification of Severe Acute Malnutrition in Infants and Children.
organic acids and acylcarnitines in biological specimens. MC, VM, SK did Geneva: World Health Organization, 2009. http://apps.who.int/iris/
and interpreted testing for potential viral causes of illness. AC, SP, and RK bitstream/10665/44129/1/9789241598163_eng.pdf (accessed
Jan 20, 2015).
did and interpreted testing for pesticide metabolites in environmental
specimens, and acetyl cholinesterase levels in human biological 17 Tong S, Chern SW, Li Y, Pallansch MA, Anderson LJ. Sensitive and
broadly reactive reverse transcription-PCR assays to detect novel
specimens. LAG, TPM, DJ, LV, KLC, and JMJ did and interpreted testing
paramyxoviruses. J Clin Microbiol 2008; 46: 2652–58.
for toxic elements and pesticide metabolites. LAH and GRT did and

8 www.thelancet.com/lancetgh Published online January 30, 2017 http://dx.doi.org/10.1016/S2214-109X(17)30035-9

 Articles

18 Tong S, Singh J, Ruone S, et al. Identification of adenoviruses in 32 Ghisla S, Melde K, Zeller HD, Boschert W. Mechanisms of enzyme
fecal specimens from wild chimpanzees (Pan trogylodytes inhibition by hypoglycin, methylenecyclopropylglycine and their
schweinfurthii) in western Tanzania. Am J Trop Med Hyg 2010; metabolites. Prog Clin Biol Res 1990; 321: 185–92.
82: 967–70. 33 Isenberg SL, Carter MD, Hayes SR, et al. Quantification of toxins in
19 Jarrett JM, Jones RL, Caldwell KL, Verdon CP. Total urine arsenic soapberry (Sapindaceae) arils: hypoglycin A and
measurements using inductively coupled plasma mass spectrometry methylenecyclopropylglycine. J Agric Food Chem 2016; 64: 5607–13.
with a dynamic reaction cell. Atomic Spectroscopy 2007; 28: 113–22. 34 Spencer PS, Palmer VS, Mazumder R. Probable toxic cause for
20 Caldwell KL, Jones R, Hollowell JG. Urinary iodine concentration: suspected lychee-linked viral encephalitis. Emerg Infect Dis 2015;
United States National Health And Nutrition Examination Survey 21: 904–05.
2001–2002. Thyroid 2005; 15: 692–99. 35 Gray DO, Fowden L. alpha-(Methylenecyclopropyl)glycine from
21 Caldwell KL, Mortensen ME, Jones RL, Caudill SP, Osterloh JD. Litchi seeds. Biochem J 1962; 82: 385–89.
Total blood mercury concentrations in the US population: 36 Sahai I, Garganta CL, Bailey J, et al. Newborn Screening for Glutaric
1999–2006. Int J Hyg Environ Health 2009; 212: 588–98. Aciduria-II: The New England Experience. JIMD Rep 2014; 13: 1–14.
22 Kuklenyik Z, Panuwet P, Jayatilaka NK, Pirkle JL, Calafat AM. 37 Meda HA, Diallo B, Buchet JP, et al. Epidemic of fatal encephalopathy
Two-dimensional high performance liquid chromatography in preschool children in Burkina Faso and consumption of unripe
separation and tandem mass spectrometry detection of atrazine and ackee (Blighia sapida) fruit. Lancet 1999; 353: 536–40.
its metabolic and hydrolysis products in urine. 38 Tanaka K, Kean EA, Johnson B. Jamaican vomiting sickness.
J Chromatogr B Analyt Technol Biomed Life Sci 2012; 901: 1–8. Biochemical investigation of two cases. N Engl J Med 1976;
23 Odetokun MS, Montesano MA, Weerasekera G, Whitehead RD Jr, 295: 461–67.
Needham LL, Barr DB. Quantification of dialkylphosphate 39 Service FJ. Hypoglycemic disorders. N Engl J Med 1995; 332: 1144–52.
metabolites of organophosphorus insecticides in human urine
40 Touma EH, Charpentier C. Medium chain acyl-CoA dehydrogenase
using 96-well plate sample preparation and high-performance liquid
deficiency. Arch Dis Child 1992; 67: 142–45.
chromatography-electrospray ionization-tandem mass spectrometry.
J Chromatogr B Analyt Technol Biomed Life Sci 2010; 878: 2567–74. 41 Barcelouex D. Chapter 3: Ackee fruit and Jamaican Vomiting
Sickness Medical Toxicology of Natural Substances. Hoboken,
24 Ellman GL, Courtney KD, Andres V, Feather-Stone RM. A new and
New Jersey: John Wiley & Sons Inc; 2008: pp 34–38.
rapid colorimetric determination of acetylcholinesterase activity.
Biochem Pharmacol 1961; 7: 88–95. 42 Lucas RM, McMichael AJ. Association or causation: evaluating links
between “environment and disease”. Bull World Health Organ 2005;
25 Lehotay SJ, de Kok A, Hiemstra M, Van Bodegraven P. Validation of
83: 792–95.
a fast and easy method for the determination of residues from
229 pesticides in fruits and vegetables using gas and liquid 43 Bandyopadhyay B, Chakraborty D, Ghosh S, et al.
chromatography and mass spectrometric detection. J AOAC Int Epidemiological Investigation of an Outbreak of Acute Encephalitis
2005; 88: 595–614. Syndrome (AES) in Malda District of West Bengal, India.
Clin Microbial 2015; 4: 181–87.
26 Isenberg SL, Carter MD, Graham LA, et al. Quantification of
metabolites for assessing human exposure to soapberry toxins 44 Biswas SK, International Centre for Diarrhoeal Diseases Research.
hypoglycin A and methylenecyclopropylglycine. Chem Res Toxicol Outbreak of illness and deaths among children living near lychee
2015; 28: 1753–59. orchards in northern Bangladesh. Bangladesh ICDDRB Health Sci Bull
2012; 10: 15–22.
27 Hoffmann G, Aramaki S, Blum-Hoffmann E, Nyhan WL,
Sweetman L. Quantitative analysis for organic acids in biological 45 Paireau J, Tuan NH, Lefrancois R, et al. Litchi-associated acute
samples: batch isolation followed by gas chromatographic-mass encephalitis in children, Northern Vietnam, 2004–2009.
spectrometric analysis. Clin Chem 1989; 35: 587–95. Emerg Infect Dis 2012; 18: 1817–24.
28 Millington DS, Roe CR, Maltby DA. Application of high resolution 46 Ministry of Health and Family Welfare Government of India.
fast atom bombardment and constant B/E ratio linked scanning to Direct Order Number A.1-4/2012/EPID/NCDC. New Delhi, 2015.
the identification and analysis of acylcarnitines in metabolic 47 Bowen-Forbes CS, Minott DA. Tracking hypoglycins A and B over
disease. Biol Mass Spectrometry 1984; 11: 236–41. different maturity stages: implications for detoxification of ackee
29 Millington DS, Terada N, Chace DH, et al. The role of tandem mass (Blighia sapida K.D. Koenig) fruits. J Agric Food Chem 2011;
spectrometry in the diagnosis of fatty acid oxidation disorders. 59: 3869–75.
Prog Clin Biol Res 1992; 375: 339–54. 48 Murphy RC, Clay KL. Synthesis and back exchange of ¹⁸O labeled
30 Rinaldo P, Cowan TM, Matern D. Acylcarnitine profile analysis. amino acids for use as internal standards with mass spectrometry.
Genet Med 2008; 10: 151–56. Biomed Mass Spectrom 1979; 6: 309–14.
31 Thompson JA, Markey SP. Quantitative metabolic profiling of
urinary organic acids by gas chromatography-mass spectrometry:
comparison of isolation methods. Anal Chem 1975; 47: 1313–21.

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