Choleragenoid

The Infectious Diseases and Beliaghata General Hospital (ID&BGH), in Kolkata, a 770 bedded hospital, provides treatment for about 20,000 to 25,000 hospitalized patients with acute diarrhoea annually. In the present systematic active surveillance, every fifth patient with diarrhoea or dysentery without other associated illness on two randomly selected days of the week was enrolled as study subjects from cases admitted at the ID&BGH. This study was conducted between November 2007 and October 2009. The dehydration status of each diarrhoea case was classified as no, some or severe dehydration according to WHO guidelines. The clinical, demographic and laboratory data was checked manually and entered into pre-designed data entry proforma developed in visual basic with inbuilt entry validation checking facilitated programme in structure query language (SQL) server by dual entry method by trained data entry professionals. Data was randomly checked and matched to derive consistency and validity for analysis. The edited data was exported and a final analysis was performed using the SPSS.17.0 software (SPSS Inc., Chicago, IL, USA).
This study was approved by the duly constituted Institutional Ethics Committee (IEC). As per the recommendation of IEC, individual informed consent was obtained from each patient enrolled in this study and confidentiality was maintained. Faecal specimens were collected in McCartney bottles using sterile catheters or as rectal swabs in Cary Blair medium and were examined within 2 hrs for 24 enteric pathogens comprising bacterial, viral and parasitic pathogens using a combination of conventional, immunological and molecular methods (Fig. 6). PCR targeting ompW and toxR were performed for the species confirmation of V. cholerae and V. fluvialis, respectively [31 (link),32 (link)]. Confirmed strains of V. parahaemolyticus, Shigella spp and Salmonella spp were serotyped using commercially available antisera (Denka Seiken, Tokyo, Japan, BioRad, Marnes-la-Coquette, France). V. cholerae strains were serotyped using antisera prepared in NICED. Representative strains of V. cholerae O1 were examined by MAMA-PCR to determine the type of cholera toxin B subunit gene (ctxB) [33 (link)]. Three different lactose-fermenting colonies isolated from each sample were picked from MacConkey agar plate and included in the multiplex PCR assay for the detection of different DEC that include enterotoxigenic E. coli (ETEC, inclusive of both heat-labile and heat-stable enterotoxin producers), enteropathogenic E. coli (typical and atypical EPEC) and enteroaggregative E. coli (EAEC) [34 (link)]. Simplex PCR was also performed for the detection of enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) [35 (link),36 (link)].
Antimicrobial susceptibility testing was performed by disk diffusion (Kirby- Bauer method) using commercially available disks (Becton Dickinson Co., Sparks, MD, USA) with interpretation stipulated by the Clinical and Laboratory Standard Institute [37 ]. Two hundred and thirty representative (one third from the total number of strains) V. cholerae O1 strains covering all the months and all the Shigella strains were included in the testing. Rotavirus was detected by polyacrylamide gel electrophoresis and silver staining [38 (link)]. Norovirus [Group I and II (NVGI and NVGII)], Sapovirus and Astrovirus were detected by RT-PCR using random primers for reverse transcription and specific primers for polymerase chain reaction [24 (link),39 (link)]. Different viruses were detected according to the appropriate amplicon sizes observed in agarose gels stained with ethidium bromide. Adenovirus was detected by the commercially available RotaAdeno VIKIA kit (biomereux, France), which is a qualitative test-based on immunochromatography in lateral flow format [40 (link)]. For detection of enteric parasites, faecal samples were processed separately for microscopic and molecular analysis. For microscopic analysis, the samples were first concentrated using formalin ethyl acetate concentration method [41 ] and an aliquot of each sample was preserved in 10% formalin and stored at 4°C for subsequent use. Aliquots of fresh stool specimens were also preserved at -80°C for ELISA and PCR assays. All the faecal samples were screened using a highly sensitive antigen capture ELISA (Tech Lab, Blacksburg, USA) and PCR for the detection of Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica. Faecal samples were processed by microscopy using iodine wet mount staining and trichome staining procedure for Blastocystis hominis [42 ].
Using the surveillance data, an estimate of the total number of cases specific for each pathogen in two consecutive years was extrapolated. From the monthly enrolled cases, the isolation rate of different pathogens was calculated for that particular month. An estimate of total number of cases with particular pathogen for a particular month was then extrapolated by multiplying the total admitted cases with particular isolation rate of the pathogenic with an assumption that similar isolation rate would be among non-enrolled cases. In this way, pathogen-specific total number of yearly estimated cases was calculated.
The risk age group was also explored for predominant enteric pathogens such as V. cholerae O1, Rotavirus, shigellae and G. lamblia by Multinomial Logistic Regression (MLR) analysis [43 (link),44 (link)]. This analysis helps to determine the likelihood age of the patient associated with any enteric pathogen. The age groups were classified into 8 categories viz. <1 year, 1-2 years, >2-5 years, >5-14 years, >14-30 years, >30-45 years, >45-60 years and >60 years and were coded from 1 to 8, respectively. Infection caused by an enteric pathogen was coded as '1' for the pathogen present and '2' for its absence. The extreme values of the classified age group was fixed as a reference category.

E15 cerebral cortices were dissociated and cultured for 2 days. Primary cultured neurons were incubated with 5 μM BODIPY‐FL C5‐LacCer (Molecular Probes) in OPTI‐MEM medium (Invitrogen) for 10 min on ice and further incubated for 30 min at 37°C. Cells were washed six times with OPTI‐MEM containing 1% BSA for 10 min at 10°C and fixed with 4% PFA in PBS for 20 min. For CTxB uptake assay, primary cultured neurons were incubated with 5 μg/ml Alexa555‐conjugated Cholera Toxin Subunit B (CTxB) (Molecular Probes) in OPTI‐MEM for 10 min on ice, washed with Neurobasal medium and further incubated with Neurobasal medium for 30 min at 37°C. After fixation with 4% PFA in PBS for 20 min, cells were subjected to immunocytochemical analyses. For transferrin uptake assay, primary cultured neurons were incubated in OPTI‐MEM media for 30 min at 37°C and treated with 20 μg/ml Alexa594‐ or Alexa555‐conjugated transferrin (Tf) (Molecular Probes) in OPTI‐MEM media. After incubation for 5–15 min on ice or for 30 min at 10°C, cultured media was replaced with preincubated neurobasal media without Alexa dye‐conjugated Tf. Subsequently, neurons were incubated for 10 or 30 min at 37°C and fixed with 4% PFA in PBS for 20 min.

Primary antibodies used in this study were anti‐Rab21 (R4405, Sigma), anti‐Rab5 (3547, Cell Signaling Technology; 46449, Cell Signaling Technology), anti‐APPL1 (3858, Cell Signaling Technology), anti‐EEA1 (07‐292, Upstate; 610456, BD Biosciences), anti‐caveolin‐1 (3238, Cell Signaling Technology for staining and immunoblotting; 610406, BD Biosciences for staining; 3267, Cell Signaling Technology for immunoblotting), anti‐Rab11 (5589, Cell Signaling Technology), anti‐Rab7 (9367, Cell Signaling Technology), anti‐Rab6 (9625, Cell Signaling Technology), anti‐Lamp1 (sc‐19992, Santa Cruz), anti‐Syntaxin‐6 (610635, BD Biosciences), anti‐Calnexin (610523, BD Biosciences), anti‐KDEL (sc‐58774, Santa Cruz), anti‐TfR (13‐6800, Invitrogen), anti‐CD44 (NB100‐65905, Novus), anti‐Endophilin‐II (sc‐365704, Santa Cruz), anti‐GFP (A‐6455, Molecular Probes; 04404‐84, Nacalai; AB16901, Millipore), anti‐mAG1 (PM052M, MBL), anti‐HA (2367, Cell Signaling Technology), anti‐N‐cadherin (C3865, Sigma; sc‐7939, Santa Cruz Biotechnology; ab98952, Abcam), anti‐Phospho Histone H3 (9701, Cell Signaling Technology), anti‐Ki67 (NCL‐Ki67p, Leica), anti‐βIII tubulin (Tuj1) (MMS‐435P, Covance), anti‐MAP2ab (ab11268, Abcam), anti‐β‐tubulin (T5201, Sigma), and anti‐β‐actin (A5441, Sigma). BODIPY‐FL C5‐LacCer (LacCer), Alexa555‐conjugated Cholera Toxin Subunit B (CTxB) and Alexa594‐ or Alexa555‐conjugated transferrin (Tf) were purchased from Molecular Probes. 4′,6‐diamidino‐2‐phenylindole dihydrochloride solution (DAPI), and Bafilomycin A1 were purchased from Wako (340‐07971) and Sigma (SML‐1661), respectively.

hDF cells (20,000 cells in 24-well plates on coverslip) were grown in complete medium for 24 h. Starvation was performed for 1 h in Hanks’ Balanced Salt solution (HBSS) before fixing with paraformaldehyde (PFA) 4% and staining with anti-LAMP1, anti-LAMP2, anti-p62, anti-LC3B, anti-TMEM175, anti-GC, and anti-GM antibodies. Briefly, incubation with blocking buffer (1 × PBS/0.1% triton/5% serum) was performed for 15 min in a humidified chamber. Primary and then secondary antibody incubations (1 × PBS/0.1% triton/antibody) were performed at 4 °C overnight and at RT for 1 h, respectively. Anti-LAMP1 (ab25630, Abcam, Cambridge, UK, 1:20), anti-LAMP2 (ab25631, Abcam, Cambridge, UK, 1:100), anti-LC3B (2775, Cell Signaling, Danvers, MA, USA, 1:200), anti-TMEM175 (19,925–1-AP, Proteintech, Manchester, UK, 1:100) and anti-p62/SQSTM1 (P0067, Sigma-Aldrich, St. Louis, MO, USA, 1:500), anti-GlcCer (RAS0010, Glycobiotech, Kuekels, Germany, 1:50) primary antibodies and anti-mouse IgG-Cy3 (C2181, Sigma-Aldrich, St. Louis, MO, USA, 1:200), anti-rabbit IgG-Cy3 (C2306, Sigma-Aldrich, St. Louis, MO, USA, 1:200), Donkey anti-Mouse IgG (H + L), Alexa Fluor™ 488 (A-21202, Thermo Fisher Scientific CA USA, 1:400), and Donkey anti-Rabbit IgG (H + L) Alexa Fluor™ 488 (A-21206, Thermo Fisher Scientific CA USA, 1:400) secondary antibodies were used. GM1 was detected through 2-h incubation with Cholera toxin subunit B (CT-B) conjugated with Alexa fluor 488 (c34775, Molecular Probes, Eugene, OR, USA, 1:50). Hoechst H3570 (Thermo Fisher Scientific, CA, USA, 1:1000) was used to visualize the nuclei. Slides were visualized with Nikon Confocal Microscope A1R and with Nikon Eclipse Ni-E Microscope.