Supplementary appendixThis appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors.

Supplement to: Shrivastava A, Kumar A, Thomas JD, et al. Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, India, 2014: a case-control study. Lancet Glob Health 2017; published online Jan 30. http://dx.doi.org/10.1016/S2214-109X(17)30035-9.

1

Association of acute toxic encephalopathy with litchi consumption in an outbreak in Muzaffarpur, 1

India, 2014: a case-control study 2

Supplementary Appendix 3

Contents 4

1.1 Evaluation of infectious pathogens .....................................................................................................................4 5

1.2 Evaluation of pesticides and heavy metals ..........................................................................................................4 6

1.3 Evaluation of pesticide residues in litchi fruit samples ........................................................................................8 7

1.4 Evaluation for hypoglycin A and MCPG ................................................................................................................9 8

1.5 Evaluation of Hypoglycin A and MCPG in litchi fruit samples ........................................................................... 10 9

1.6 Figure of Electroencephalogram of child with hypoglycemic encephalopathy, Muzaffarpur, India. ............... 10 10

1.7 Unmatched bivariate analysis of case control study, controlling for age ......................................................... 11 11

References for Supplementary Appendix: .............................................................................................................. 13 12

13 14

15

2

Aakash Shrivastava, PhD1, Anil Kumar, MD1, Jerry D. Thomas, MD2, Kayla F. Laserson, ScD3,13, Gyan 16

Bhushan, MD3, Melissa D. Carter, PhD2, Mala Chhabra, MD1, Veena Mittal, MD1, Shashi Khare, MD1, 17

James J. Sejvar, MD4, Mayank Dwivedi, MD3, Samantha L. Isenberg, PhD6, Rudolph Johnson, PhD2, 18

James L. Pirkle, MD2, Jon D. Sharer, PhD7, Patricia L. Hall, PhD7, Rajesh Yadav, MBBS8, Anoop 19

Velayudhan, MBBS8, Mohan Pappanna, MD8, Pankaj Singh, MBBS8, D. Somashekar, MD8, Arghya 20

Pradhan, MBBS8, Kapil Goel, MD8, Rajesh Pandey, MBBS8, Mohan Kumar, MBBS8, Satish Kumar, MD8, 21

Amit Chakrabarti, MD9, P. Sivaperumal, PhD9, A. Ramesh Kumar PhD9, Joshua G. Schier, MD2, Arthur 22

Chang, MD2, Leigh Ann Graham, PhD6, Thomas P. Mathews, PhD6, Darryl Johnson, PhD10, Liza Valentin, 23

PhD2, Kathleen L. Caldwell, PhD2, Jeffery M. Jarrett, MS2, Leslie A. Harden, MS11, Gary R. Takeoka, 24

PhD11, Suxiang Tong, PhD12, Krista Queen, PhD12, Clinton Paden, PhD12, Anne Whitney, PhD4, Dana L. 25

Haberling, MSPH4, Ram Singh, PhD 1, Ravi Shankar Singh, MD1, Kenneth C. Earhart, MD3,13, A.C. 26

Dhariwal, MD14, L.S. Chauhan, DPH1, S. Venkatesh, MD1, Padmini Srikantiah, MD3,13 27

28

1National Centre for Disease Control, India, Directorate General of Health Services, Ministry of Health 29

and Family Welfare, Government of India, 22 Sham Nath Marg, Delhi 110054, India 30

2National Center for Environmental Health, U.S. Centers for Disease Control and Prevention, 4770 31

Buford Highway Building 109, Atlanta, Georgia 30341, USA 32

3Global Disease Detection Program - India, U.S. Centers for Disease Control and Prevention, Embassy of 33

the United States, Shanti Path, Chanakyapuri, New Delhi, 110021 India 34

4Muzaffarpur District Health Department, Government of Bihar, Sadar Hospital, Muzaffarpur, Bihar 35

842001, India 36

5National Center for Emerging and Zoonotic Infectious Diseases, U.S. Centers for Disease Control and 37

Prevention, 1600 Clifton Road, Atlanta, Georgia, 30333 USA 38

3

6Battelle at the Centers for Disease Control and Prevention, 4770 Buford Highway Building 109, 39

Atlanta, GA, 30341 USA 40

7Emory Genetics Laboratory, Department of Human Genetics, Emory University, 2165 North Decatur 41

Road, Decatur, Georgia, 30033 USA 42

8India Epidemic Intelligence Service, National Centre for Disease Control, India, Directorate General of 43

Health Services, Ministry of Health and Family Welfare, Government of India, 22 Sham Nath Marg, 44

Delhi, 110054 India 45

9National Institute of Occupational Health, Indian Council of Medical Research, Ministry of Health and 46

Family Welfare, Government of India, Meghani Nagar, Ahmedabad, Gujarat 380016, India 47

10Oak Ridge Institute for Science and Education Fellow at the Centers for Disease Control and 48

Prevention, 4770 Buford Highway Building 109, Atlanta, GA, 30341 USA 49

11Western Regional Research Center, United States Department of Agriculture, 800 Buchanan Street 50

Albany, California 94710 USA 51

12National Center for Immunizations and Respiratory Diseases, U.S. Centers for Disease Control and 52

Prevention, 1600 Clifton Road, Atlanta, Georgia 30333, USA 53

13Center for Global Health, U.S Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, 54

Georgia, 30333 USA 55

14National Vector Borne Disease Control Programme, Directorate General of Health Services, Ministry 56

of Health and Family Welfare, Government of India, Nirman Bhavan, New Delhi,110011 India 57

58

Corresponding author: 59

Padmini Srikantiah, MD MPH, US Centers for Disease Control and Prevention 60

Email: pks6@cdc.gov, Telephone: +91-11-2419-8678 61

4

1.1 Evaluation of infectious pathogens 62

As previously reported 1, CSF specimens were examined at the Indian National Centre for Disease 63

Control (NCDC) for Japanese encephalitis virus (JEV) and West Nile virus (WNV) using polymerase chain 64

reaction (PCR) methods. A subset of CSF specimens was examined using a multiplex PCR platform 65

assay with the capacity to detect 11 viruses: Herpes simplex viruses 1 and 2, human herpes viruses 6 66

and 7, cytomegalovirus, varicella zoster virus, Epstein-Barr virus, parechovirus, adenovirus, 67

enteroviruses, and parvovirus B19. 68

69

CSF and serum specimens evaluated at the U.S. Centers for Disease Control and Prevention (CDC) 70

pathogen discovery laboratory were extracted with Qiagen One For All Kit to yield total nucleic acids 71

(TNA). TNA from each specimens was screened for the presence of viral pathogens (adenoviruses, 72

alphaviruses, arenaviruses, bornaviruses, bunyaviruses, coronaviruses, flaviviruses, influenza viruses, 73

paramyxoviruses, parvoviruses, piconaviruses, polyomaviruses, reoviruses, and rhabdoviruses) by their 74

generic pan-viral family/genus PCRs, and bacterial pathogens by 16s rDNA PCR. All PCRs reactions were 75

performed and analyzed according to previously described protocols 2, 3. TNA from 40 CSF specimens 76

was also pooled by every 10 specimens and subjected to high-throughput sequencing analysis using 77

MiSeq (Illumina). 78

1.2 Evaluation of pesticides and heavy metals 79

To screen for organophosphate exposure (Table 1), case patient urine specimens were analyzed at CDC 80

by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) for 6 81

organophosphate pesticide metabolites: dimethylphosphate, diethylphosphate, 82

dimethylthiophosphate, dimethyldithiophosphate, diethylthiophosphate, and diethyldithiophosphate 83

5

(Table 2) 4. ID-LC-MS/MS assays were also performed on case urine samples to assess for atrazine and 84

six of its metabolites: atrazine mercapturate, desethyl atrazine mercapturate, diaminochlorotriazine, 85

desethyl atrazine, deisopropyl atrazine and deisopropyl atrazine mercapturate (Table 3) 5. Estimation 86

of RBC acetylcholinesterase and plasma butyrylcholinesterase activity was conducted on blood 87

samples of cases and controls utilizing the Ellman method 6, at the National Institute of Occupational 88

Health (NIOH), India. 89

90

6

Table 1: Pesticides Associated with Specific Dialkyl Phosphates 91 92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

Table 2: Urine Dialkylphosphates 108

Analyte

LOD

N

N of detects

Geometric Mean

Average Min Max

Diethyldithiophosphate 0.50 µg/L 80 0 <LOD <LOD <LOD <LOD

Diethylphosphate 0.10 µg/L 80 18 * * <LOD 7.4

Dimethyldithiophosphate 0.10 µg/L 80 3 * * <LOD 0.90

Dimethylphosphate 0.50 µg/L 79** 58 1.63 4.88 <LOD 116

Dimethylthiophosphate 0.10 µg/L 79** 17 * * <LOD 2.02

Diethylthiophosphate 0.25 µg/L 80 2 * * <LOD 0.89

* GM and Average not calculated for detection rate <60% 109 **Interfering substance present in one sample. 110

Pesticide

(CAS number)

Dimethyl-

phosphate

(813-79-5)

Dimethylthio-

phosphate

(1112-38-5)

Dimethyldithio-

phosphate

(756-80-9)

Diethyl-

phosphate

(598-02-7)

Diethylthio-

phosphate

(2465-65-8)

Diethyldithio-

phosphate

(298-06-6)

Azinphos methyl

Chlorethoxyphos

Chlorpyrifos

Chlorpyrifos methyl

Coumaphos

Dichlorvos (DDVP)

Diazinon

Dicrotophos

Dimethoate

Disulfoton

Ethion

Fenitrothion

Fenthion

Isazaphos-methyl

Malathion

Methidathion

Methyl parathion

Naled

Oxydemeton-methyl

Parathion

Phorate

Phosmet

Pirimiphos-methyl

Sulfotepp

Temephos

Terbufos

Tetrachlorvinphos

7

Table 3: Atrazine and Metabolites 111 112

Analyte

LOD

N

Average

Atrazine 0.50 µg/L 80 <LOD

Atrazine mercapturate 0.50 µg/L 80 <LOD

Desethyl atrazine mercapturate 0.10 µg/L 80 <LOD

Diaminochlorotriazine 0.50 µg/L 80 <LOD

Desethyl atrazine 0.25 µg/L 80 <LOD

Desisopropyl atrazine 0.25 µg/L 80 <LOD

Desisopropyl atrazine mercapturate

0.10 µg/L 80 <LOD

All samples measured < LOD 113

A panel of whole blood metals (cadmium, manganese, mercury, lead, and selenium) was performed on 114

case patient specimens using inductively coupled dynamic reaction cell plasma mass spectrometry 115

(Table 4) (ICP-DRC-MS)7. Total urine arsenic along with a panel of urine metals (antimony, barium, 116

beryllium, cadmium, cesium, cobalt, lead, manganese, molybdenum, platinum, strontium, thallium, tin, 117

tungsten, and uranium) was performed on patient specimens using ICP-DRC-MS 8, 9 (Table 5). 118

119

Table 4: Blood levels of heavy metals of Muzaffarpur case-patients conducted at CDC, 2014 120

121

Analyte Units LOD N Geometric Mean

Average Min Max

Cadmium µg/L 0.10 75* 0.28 0.33 <LOD 1.10

Manganese µg/L 0.99 75 19.0 21.1 7.3 57

Lead µg/dL 0.07 75 3.85 4.77 0.55 15

Selenium µg/L 24 75 120 123 71 230

Total Mercury µg/L 0.28 75 0.80 0.90 0.32 2.6

122 *10 BCD results <LOD were replaced with sqrt(LOD)/2. 123 124 125

126

127

8

Table 5: Urine elemental and elemental species results 128 129

Analyte LOD

(µg/L) N*

Geometric Mean

Average Min Max

AsT 0.26 77 11.5 21.6 0.51 130

As3 0.12 10 3.23 3.48 1.8 6.4

As5 0.79 10 1.18 1.22 0.92 2.1

AsB 1.16 10 <LOD <LOD <LOD <LOD

AsC 0.11 10 <LOD <LOD <LOD <LOD

DMA 1.91 10 49.7 52.9 32 89

MMA 0.2 10 1.59 1.91 1.0 5.3

TMO 0.17 10 <LOD <LOD <LOD <LOD

Ba 0.06 77 3.55 16.6 0.18 350

Be 0.016 77 0.057 0.059 <LOD 0.064

Cd 0.036 76 0.225 1.11 <LOD 29

Co 0.023 77 0.673 1.38 0.11 25

Cs 0.086 77 4.56 5.91 0.52 25

Mn 0.13 76 1.55 11.4 <LOD 160*

Mo 0.8 77 20.3 33.1 1.3 140

Pb 0.03 77 2.14 5.27 0.20 100

Pt 0.01 77 0.03 0.04 <LOD 0.053

Sb 0.022 77 0.118 1.39 <LOD 76

Sn 0.09 76 10.7 46.2 0.27 570*

Sr 2.3 76 54.6 108 2.5 710*

Tl 0.018 77 0.097 0.128 <LOD 0.63

W 0.018 77 0.265 0.547 <LOD 4.9

U 0.002 77 0.043 0.154 0.002 3.3

130 *Results <LOD were replaced with sqrt(LOD)/2. Three results were not reportable because they were above the 131 reportable range of the method for Mn (1 result >200 ug/L), Sn (1 result > 600 ug/L), and Sr (1 result > 1200 132 ug/L). One Cd result was not reportable due to the presence of an interfering substance (elevated Sn). Only 10 133 urine samples near or above 50 µg/L total arsenic were analyzed for arsenic species. 134 135

1.3 Evaluation of pesticide residues in litchi fruit samples 136

Collected litchi fruit samples were analyzed at NIOH for 2,4-D Ethyl Ester, acephate, acetamicprid, 137

alphamethrin, atrazin, butachlor, carbandazim, carbofuran, chlorpyriphos, cypermethrin, deltamethrin, 138

dichlorovos, dicofol, hexaconazole, imidacloprid, malathion, methyl parathion, metribuzin, 139

monocrotophos, pendimethalin, phorate, pretilachlor, profenofos, propargite, thiomethoxan and 140

triazophos using the Quick Easy Cheap Effective Rugged and Safe method 10 for the analysis of food 141

samples. 142

9

1.4 Evaluation for hypoglycin A and MCPG 143

Evaluation of urine metabolites of hypoglycin A and MCPG 144

Details regarding the experimental procedure and method used for the evaluation of hypoglycin A and 145

MCPG urinary metabolites have been previously published11. Briefly, urine specimens were processed 146

by isotope dilution (20X), separated by high pressure liquid chromatography coupled to tandem mass 147

spectrometry (HPLC-MS/MS). Isotopically-labeled internal standards were used for quantitation of the 148

hypoglycin A and MCPG urine metabolites. The toxins were excreted in urine as glycine conjugates 149

known as methylenecyclopropylacetyl glycine (MCPA-Gly) for hypoglycin A and 150

methylenecyclopropylformyl glycine (MCPF-Gly) for MCPG. Quantitation was completed between the 151

linear range of 0.100-20.0 µg/mL and corrected by creatinine for reportable values of µg of metabolite 152

per mg of creatinine. 153

154

Evaluation of plasma acylcarnitines 155

Acylcarnitines in plasma samples were extracted into a mixture of methanol and isotope-labeled 156

internal standards (Cambridge Isotope Laboratories) 12-14. The solvent was then evaporated under a 157

stream of nitrogen, and the residual acylcarnitines and internal standards were converted to butyl 158

esters via heated incubation in butanolic HCl. Flow injection ESI-MS/MS analysis of the butylated 159

acylcarnitines was performed using a precursor ion scan, specifically for the detection of a common 160

m/z = 85 amu fragment generated by collision induced dissociation with argon. Quantification of 161

individual acylcarnitine species was afforded by comparison with selected internal standards. 162

163

Evaluation of urinary organic acids 164

10

Organic acids in urine (equivalent to 1 µmol of creatinine) were acidified, oximated, and extracted with 165

diethyl ether/ethyl acetate 15-17. The organic phase was dried under nitrogen prior to preparing stable 166

trimethylsilyl (TMS) derivatives for gas chromatography mass spectrometry (GC-MS) analysis with 167

electron ionization MS. Quantification of selected organic acids was accomplished by individual 168

calibration standards with internal standard correction. Additional organic acids and urine metabolites 169

were identified qualitatively based on comparison of retention time and MS spectra to a library 170

consisting of both commercially available compounds, and internally identified metabolic 171

intermediates. 172

1.5 Evaluation of Hypoglycin A and MCPG in litchi fruit samples 173

Details regarding the experimental procedure and method used for the evaluation of hypoglycin A and 174

MCPG in soapberry arils have been published separately18. Briefly, homogenate arils were dehydrated 175

for 1 hour at 57 °C followed by homogenization in 80% ethanol. The supernatant was dried under 176

nitrogen at 60 °C and resuspended in water at a concentration of 2 mg dried fruit per mL of water. 177

Each extract was derivatized with dansyl chloride followed by a solid-phase extraction (SPE) with a 178

Waters HLB 96-well SPE plate. The SPE eluent was dried under nitrogen at 60 °C and resuspended in 179

0.1 % formic acid in water. The samples were then analyzed by HPLC-MS/MS and reported as µg/g of 180

dried aril. 181

182

1.6 Figure of Electroencephalogram of child with hypoglycemic encephalopathy, Muzaffarpur, India. 183 184 Figure: Electroencephalogram of child with hypoglycemic encephalopathy, Muzaffarpur, India. Tracing 185 demonstrates left posterior quadrant discharges followed by generalization 186 187

11

188 189

1.7 Unmatched bivariate analysis of case control study, controlling for age 190

Unmatched bivariate analyses, controlled for age, were conducted to assess for potential exposures 191

associated with illness. Age was adjusted for as a continuous variable in logistic regression. The 192

calculated odds ratios are listed below and were similar to what was observed in matched bivariate 193

analyses. 194

Table 6: Exposures associated with illness in unmatched bivariate analysis of case control study, 195

controlling for age, Muzaffarpur, June – July 2014 196

KEY EXPOSURES CASES CONTROLS OR

12

(N=104) (N=104) (95% CI)

Ate litchi* 67/103 (65%) 23/102 (23%) 6.5 (3·5 -12.1)

Visited fruit orchard* 52/100 (52%) 18/98 (32%) 5·0 (2·6 –9.6)

Parent visited fruit orchard* 29/95 (31%) 16/99 (16%) 2·3 (1·2 – 4·6)

Absence of evening meal* 76/98 (78%) 51/88 (58%) 2·6 (1·4 – 5.1)

Socioeconomic Index below

poverty line

57/104(55%) 49/104 (47%) 1·4 (0·8 – 2·5)

Routinely wash vegetables

and fruits

32/99 (32%) 58/83 (70%) 0·20 (0·1 – 0·4)

*in 24 hours prior to symptom onset 197

198

199 200

13

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