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Title: Water, sanitation and hygiene predictors of diarrhoeal infection for rural and urban communities in south India among children and adults
Authors: D. Kattula, M. R. Francis, A. Kulinkina, R. Sarkar, V. M. Raghava, S. Babji, H. D. Ward, G. Kang, V. Balraj, E. N. Naumova
Supplementary Material
Supplementary Document S1: Laboratory methods for testing the diarrhoeal stool samples for the presence of viral, bacterial and parasitic pathogens.
Bacterial culture:
Stool samples were subjected to standard bacteriology culture methods to identify bacterial pathogens. Samples were plated on MacConkey agar, Xylose Lysine Deoxycholate agar and Thiosulfate Citrate Bile salts Citrate agar ADDIN EN.CITE Forbes200776(1, 2)76766Forbes, B. A.Sahm, D. F.Weissfeld, S.Bailey & Scott's Diagnostic Microbiology12th2007St. LouisMosbyMurray19997373736Murray, P. R.Baron, E. J.Pfaller, M. A.Tenover, F. C.Manual of Clinical Microbiology7th1999ASM Press9781555813710http://books.google.co.in/books?id=Ys77LwEACAAJ( HYPERLINK \l "_ENREF_1" \o "Forbes, 2007 #76" 1, HYPERLINK \l "_ENREF_2" \o "Murray, 1999 #73" 2). After overnight incubation at 37C, the suspect colonies were further characterized by using the API-20E enteric identification system and serological confirmation using specific antisera (Denka Seiken Co.Ltd., Japan).
Multiplex polymerase chain reaction (PCR) to detect diarrheagenic E.coli pathotypes:
Three to five similar looking lactose fermenting colonies from MaConkey agar were used to extract bacterial DNA by heating them at 100C in a water bath. Multiplex PCR was performed to determine the diarrheagenic E.coli pathotypes - Enteroaggregative E.coli, Enteroinvasive E.coli, Enteropathogenic E.coli, Enterotoxigenic E.coli and Enterohemorrhagic E.coli ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_2" \o "Murray, 1999 #73" 2-4).
Stool microscopy:
Stool samples were concentrated by formalin ether concentration technique and parasitic ova and cyst were identified by wet preparation. Further, modified acid fast staining was performed to look for acid fast opportunistic parasitic pathogens ADDIN EN.CITE Ash198779(5)79796Ash, L. R.Orihel, T. C. Parasites, a Guide to Laboratory Procedures and Identification1987ChicagoAmerican Society of Clinical Pathologists( HYPERLINK \l "_ENREF_5" \o "Ash, 1987 #79" 5).
Stool antigen detection ELISA for rotavirus:
Commercially available, antigen detection ELISA kits were used to detect rotavirus (genogroup A) as per kit protocol ADDIN EN.CITE Scientific201477(6, 7)777712Thermo ScientificProspecT Rotavirus Microplate assay2014http://www.fisher.be/content/dam/tfs/SDG/MBD/MBD%20Documents/Instructions%20For%20Use/ProSpecT/Rotavirus%20Microplate%20Assay/X7596C.pdf2014Aug 21Scientific201478787812Thermo ScientificProSpecT Campylobacter Microplate Assay2014Aug 212014http://www.fisher.be/content/dam/tfs/SDG/MBD/MBD%20Documents/Instructions%20For%20Use/ProSpecT/Campylobacter%20Microplate%20Assay/X7595A.pdf( HYPERLINK \l "_ENREF_6" \o "Scientific, 2014 #77" 6).
PCR to detect norovirus:
Semi quantitative real time PCR was performed on RNA extracted from stool samples to detect Norovirus GI and GII using previously published primers ADDIN EN.CITE ADDIN EN.CITE.DATA ( HYPERLINK \l "_ENREF_8" \o "Kageyama, 2003 #80" 7).
References:
ADDIN EN.REFLIST (1) Forbes BA, Sahm DF, Weissfeld S. Bailey & Scott's Diagnostic Microbiology, 12th edn. St. Louis: Mosby, 2007.
(2) Murray PR, et al. Manual of Clinical Microbiology. 7th edn. Washington, D.C.: ASM Press, 1999.
(3) Luscher D, Altwegg M. Detection of shigellae, enteroinvasive and enterotoxigenic Escherichia coli using the polymerase chain reaction (PCR) in patients returning from tropical countries. Molecular and Cellular Probes 1994; 8: 285-290.
(4) Nguyen TV, et al. Detection and characterization of diarrheagenic Escherichia coli from young children in Hanoi, Vietnam. Journal of Clinical Microbiology 2005; 43: 755-760.
(5) Ash LR, Orihel TC. Parasites, a Guide to Laboratory Procedures and Identification. Chicago: American Society of Clinical Pathologists, 1987.
(6) Thermo Scientific. ProspecT Rotavirus Microplate assay. 2014. [http://www.fisher.be/content/dam/tfs/SDG/MBD/MBD%20Documents/Instructions%20For%20Use/ProSpecT/Rotavirus%20Microplate%20Assay/X7596C.pdf]. Date accessed: 21-Aug-2014
(7) Kageyama T, et al. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. Journal of Clinical Microbiology 2003; 41: 1548-1557.
Supplementary Table S2: Coliform counts of water samples obtained from the taps (source) and households (point-of-use)
TAPSHOUSEHOLDSTotal coliformFaecal coliformTotal coliformFaecal coliformRural Urban RuralUrbanRuralUrbanRuralUrbanNumber of samples tested697353698346536596526610Minimum count3322451822Maximum count281002740021702980298002980029802950Mean (SD) count4130 (4295)4553 (5170)184 (273)290 (466)5721 (5699)6701 (6347)366 (617)431 (645)Median (IQI) count2600
(900 - 5800)2700
(800 - 6600)72
(25 - 270)150
(31 - 360)4200
(1700 - 7700)4850
(1900 - 9050)151
(34 - 380)220
(43 - 430) SD = Standard Deviation
IQI = Inter-quartile interval
Supplementary Table S3: Age wise distribution of enteric pathogens isolated from the diarrhoeal stool samples of study participants
PathogenAge categories (at recruitment)< 12 months*12-24 months*25-59 months* e" 6 0 m o n t h s * N u m b e r o f s a m p l e s 4 1 8 6 5 2 3 0 G i a r d i a 2 ( 5 ) 9 ( 1 0 ) 1 2 ( 2 3 ) 4 ( 1 3 ) C r y p t o s p o r i d i u m 1 ( 2 ) 1 ( 1 ) 4 ( 8 ) 0 ( 0 ) A s c a r i s 0 ( 0 ) 0 ( 0 ) 2 ( 4 ) 1 ( 3 ) R o t a v i r u s 3 ( 7 ) 5 ( 6 ) 1 ( 2 ) 0 ( 0 ) S h i g e l l a 2 ( 5 ) 3 ( 3 ) 2 ( 4 ) 1 ( 3 ) V i b r i o 0 ( 0 ) 1 ( 1 ) 0 ( 0 ) 0 ( 0 ) H. nana0 (0)1 (1)0 (0)1 (3)Entamoeba histolytica0 (0)0 (0)0 (0)1 (3)Total diarrhoeagenic E. Coli 10 (24)24 (28)11 (21)4 (13) EAEC8 (20)9 (10)3 (6)1 (3) ETEC0 (0)6 (7)4 (8)3 (10) EIEC2 (5)3 (3)1 (2)0 (0) EPEC1(2)8 (9)4 (8)1 (3)
* Numbers in parenthesis are percentages
More than one E. coli pathotype identified in 4 samples, hence the sum of individual pathotypes is greater than the total of number of diarrhoeagenic E .coli positive samples
Supplementary Table S4: Univariate regression analysis for risk factors of diarrhea and duration of diarrhea at household level
Variable RURALURBANFamily diarrheal episodesDuration of diarrhea Family diarrheal episodesDuration of diarrhea n (%)IRR (95% CI)IRR (95% CI)n (%)IRR (95% CI)IRR (95% CI)Person to Person transmissionPresence of siblings under 5yrs old40 (28.5)3.46 (2.72-4.41)*3.61 (2.46 -5.28)*35 (21.8)1.95 (1.52 -2.50)*2.90 (1.78-4.71)*Crowding (More than 5 individuals per room)5 (4-6)2.11 (1.15-3.89)*2.27 (1.42 -3.62)*5 (4-6)1.31 (0.21-2.02)1.83 (1.81-1.84)*Presence of family members over 60 yrs old49 (35.0)1.68 (0.84-3.35)1.82 (1.06-3.14)22 (13.7)1.03 (0.59-1.77)0.81 (0.43-1.55)Socio economic indicatorsLow SES105 (75.0)1.42 (1.00 -2.03)*1.27 (0.87-1.85)124 (77.5)1.37 (0.91-2.07)2.07 (1.27-3.38)*Kutcha house19 (13.5)3.05 (0.75-12.44)3.24 (0.81-12.91)24 (15.0)1.06 (0.90-1.23)1.67 (1.22-2.29)Illiterate head of the household21 (15.0)1.10 (0.46-2.60)1.20 (0.45-3.21)38 (23.7)1.19 (1.06-1.34)1.27 (0.75-2.14)One room house44 (31.4)3.02 (1.53-5.95)*2.61 (1.11-6.15)74 (46.2)1.06 (0.81-1.39)1.06 (0.75-1.49)Firewood for cooking79 (56.4)1.34 (0.56-3.20)1.35 (0.46-3.95)18 (11.2)0.80 (0.57-1.13)0.85 (0.57-1.26)Animal to Human transmissionPresence of animal at home86 (61.4)2.46 (0.65-9.28)2.59 (0.54 -12.45)28 (17.5)0.70 (0.46-1.07)1.04 (0.85-1.26)Presence of flies around home134 (95.7)0.78 (0.18-3.37)0.94 (0.27-3.26)123 (76.8)0.92 (0.44-1.94)0.76 (0.18-3.09)Animal shed in/around house80 (57.1)1.23 (0.48-3.15)1.24 (0.54-2.83)24 (15.0)0.69 (0.48-0.98)0.84 (0.33- 2.13)Use of cow dung in the household126 (90.0)0.80 (0.16-3.89)0.63 (0.11-3.56)Handle waste with bare hands94 (74.6)0.64 (0.19-2.07)0.47 (0.14-1.59)Hygienic PracticesAlways purify water17 (12.1)1 (0.36-2.77)0.72 (0.34-1.51)105 (65. 6)0.79 (0.41-1.52)0.84 (0.65-1.08)Washing fruits and vegetable with drinking water 16 (11.4)0.32 (0.05-1.95)0.26 (0.04-1.43)55 (34.4)0.69 (0.53-0.89)*0.75 (0.37-1.49)Waste DisposalDisposal in dust bin/ dug pits38 (27.1)0.99 (0.31-3.12)1.10 (0.40-3.03)110 (68.8)1.12 (0.65-1.91)1.43 (0.73-2.77)SanitationPresence of latrine at house81 (57.8)0.55 (0.31- 0.98)0.55 (0.32-0.95)Use of latrine for defecation63 (45.0)0.48 (0.38-0.60)*0.48 (0.45-0.52)*Regular water in the latrine 17 (20.9)0.63 (0.48-0.83)*0.73 (0.63-0.86)*57 (36.5)0.66 (0.37-1.17)0.79 (0.25-2.45)Wash hands with soap and water53 (37.8)0.69 (0.27-1.76)0.69 (0.27-1.76)99 (61.9)1.40 (0.65-3.02)1.41 (0.65-3.02)* Significant (p<0.05)
Supplementary Table S5: Univariate regression analysis for risk factors of diarrhea and duration of diarrhea in index children
VariableRURALURBAN Diarrheal episodesDuration of diarrhea Diarrheal episodesDuration of diarrhean (%)IRR (95% CI)IRR (95% CI)n (%)IRR (95% CI)IRR (95% CI)Person to Person transmissionPresence of siblings under 5yrs old 40 (28.6)3.17 (2.19-4.57)*3.02 (1.77-5.15)*35 (21.88)2.02 (1.16-3.15)*3.24 (1.83-5.74)*Crowding (more than 5 individuals per room)5 (4-6)1.70 (0.66-4.36)1.57 (0.83-2.98)5 (4-6)1.36(0.98-1.87)2.07 (1.96-2.18)*Presence of family members over 60 yrs old49 (35)1.46 (0.46-4.631.39 (0.51-3.78)22 (13.8)1.10(0.45-2.64)0.75 (0.32-1.76)Female81 (57.8)1.06 (0.60-1.86)1.35 (0.76-2.41)83 (51.8)0.83 (0.75-0.93)*1.10 (0.83-1.45)Socio economic indicatorsLow SES105 (75)0.95 (0.72-1.25)0.87 (0.66-1.14)124 (77.5)1.24 (1.01-1.51)*1.89 (1.50-2.39)*Kutcha house19 (13.6)3.26 (0.93-11.34)*3.41 (1.12-10.36)* 24 (15)1.10 (0.74-1.64)1.79 (0.87-3.69)Illiterate head of the household21 (15)1.02 (0.64-1.60)1.06 (0.60-1.92)38 (23.8)1.22 (0.84-1.77)1.36 (0.46-3.98)one room house44 (31.4)3.18 (2.08-4.86)* 2.55 (1.33-4.88)*74 (46.3)1.08 (0.82-1.41)0.94 (0.72-1.21)Firewood for cooking79 (56.43)1.39 (0.77-2.53)1.54 (0.72-3.27)18 (11.3)0.86 (0.65-1.14)1.00 (0.74-1.36)Animal to Human transmissionPresence of animal at home86 (61.4)3.48 (1.86-6.25)*4 .02 (1.66-9.71)*28 (17.5)0.56 (0.40-0.77) *0.58 (0.46-0.74)*Presence of flies around home134 (95.7)1.27 (0.13-11.9)1.83 (0.22-14.92)123 (76.9)0.78 (0.35-1.74)0.63 (0.14-2.69)Animal shed in/around house80 (57.1)1.70 (0.96-3.01)*1.61 (1.12-2.31)*24 (15)0.52 (0.44-0.62)*0.46 (0.38-0.57)*Physical contact with animals35 (25)1.42 (0.86-2.35)1.2 (0.58-2.44)19 (12)0.44 (0.20-0.97)*0.40 (0.21-0.78)*Use of cow dung in the household126 (90)0.75 (0.65-0.85)*1.16 (0.22-6.09)Handle waste with bare hands94 (74.6)0.71 (0.24-2.07)0.57 (0.17-1.83)Hygienic PracticesAlways purify water17 (12.1)1.13 (0.39-3.24)0.83 (0.38-1.82)105 (65. 6)0.79 (0.49-1.27)0.78 (0.60-1.00)Boil drinking water28 (20)0.62 (0.37-1.05)0.54 (0.29-1.00)37 (23.1)0.86 (0.85-0.87)*0.82 (0.73-0.92)*Washing fruits and vegetable with drinking water 16 (11.4)2.41 (0.36-15.76)3.12 (0.52-18.74)55 (34.4)1.36 (1.09-1.71)*1.25 (0.62-2.53)Waste DisposalDisposal in dust bin/ dug pits38 (27.1)0.68 (0.31 -1.46)0.80 (0.47 -1.36)110 (68.75)1.10 (0.56 -2.19)1.33 (0.53-3.32)SanitationPresence of latrine at house81 (57.9)0.49 (0.38 - 0.64)*0.47 (0.36 - 0.63)*Open field defecation87 (62.1)2.38 (1.58 - 3.58)*2.23(1.46 - 3.40)*92 (57.50) $ @ O Y 1 3 I J K l[lL h
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