Thimerosal

In the study, 104 children (52 male and 52 female) from the ongoing malnutrition and enteric diseases (MAL-ED) study were randomly included. The study protocol was approved by the institutional review board of icddr,b. The approved protocol number was PR 2008–020. An informed consent paper was signed by each participant or his/her guardians. The children were apparently healthy. Any children suffering from any diarrheal or fever illnesses, congenital defect were excluded from this study. Median age of the children that participated was 15.0 months (range 6.9 to 25.8 months). Fasted children (at least 2 hours) were instructed to drink a solution containing lactulose (250 mg/ml, Osmolax, Square Pharmaceutical Ltd, Dhaka, Bangladesh) and mannitol (50 mg/ml, Sigma, St. Louis, MO, USA) in a dose of 2 ml/Kg of body weight, or a maximum 20 ml. The children were requested to empty their bladders before ingesting the test sugar solution. A pediatric urine collection bag (Fisher cat. #22275347 or Hollister U-bag #7501) was attached to the children to collect their urine. The children were allowed to return to their regular diet 30 minutes after ingestion of LM test solution and after 2 hours, 2 ml of urine were removed from collection bag; then collection was continued up to 5 hours. After 2 and 5 hours, total urine volume was measured and recorded. 1–2 drops of thimerosal (Sigma Aldrich) were added and samples were stored in a -80°C freezer until lactulose and mannitol concentration could be determined independently by HPAE employing pulsed amperometric detection (PAD) and LC-MSMS platform.

Silvio X10/7, Y, M6241, ERA and CLBrenerLuc trypomastigotes were incubated at 37°C 5% CO₂ with 1.6×10⁷ Vero cells overnight at MOI10 in a T225 flask. Extracellular parasites were removed by aspirating cell culture supernatant, and washing Vero monolayer with 10 ml serum-free DMEM three times. Vero cells were then harvested by trypsinization and dispensed at 2×10³ per well (50 μl in DMEM/ 1% FCS) into 384 well plates pre-stamped with drug in DMSO using automated washer/ dispenser EL406 and liquid handling software (Biotek). Plates were then incubated at 37°C in 5% CO₂ for 72, 96 and 120 h.
Plates were subsequently fixed with 4% formaldehyde for 20 min, permeabilised and stained with 5 μgml^-1 Hoechst 33342/ 0.1% Triton/ PBS Thimerosal 20 min. Automated imaging was performed by the Operetta high content imaging system using 20x objective (PerkinElmer). Images were analysed with an algorithm generated in Columbus (PerkinElmer) to segment Vero cell nuclei, Vero cell cytoplasm and parasite nuclei/ kinetoplasts, reporting number of amastigotes per Vero cell and total number of Vero cells. Compound potencies against T. cruzi parasites were calculated in IDBS Activitybase using total number of amastigotes per well and Vero toxicity curves generated using number of host cells. All data was normalised to percent inhibition based on the raw data values for the 100% effect control (50 μM nifurtimox) and the 0% effect control (DMSO) on each plate at each time point. Curve fitting was carried out using a four-parameter equation as previously described [68 (link)].

5 μg/ml monoclonal anti-Aβ34 (226), anti-Aβ40 (G2-10) or anti-Aβ42 (G2-13) capture antibody in 100 mM sodium carbonate (pH 9.6) were used to coat the 96-well Nunc™ plates (Thermo Fisher Scientific Inc.). The sealed plates were incubated overnight at 4 °C with gentle shaking. Plates were washed 5 times 10 min with PBS-T washing buffer (1.1 mM NaH₂PO₄, 8.5 mM Na₂HPO₄, 13.7 mM NaCl, (pH 7.4), 0.1% Tween-20). 250 μl Stabil Coat®Immunoassay Stabilizer (SurModics Inc.) was used for blocking and plates were incubated for 2 h at room temperature with gentle shaking. 50 μl of 0.075 μg/ml detection antibody, W02-biotin, in assay buffer (90% 11 mM NaH₂PO₄, 85 mM Na₂HPO₄, 137 mM NaCl, (pH 7.4), 0.5% Tween-20, 1.5% BSA, 0.01% Thimerosal, and 10% SeaBlock blocking buffer (Thermo Fisher Scientific Inc.) was loaded to the wells together with 50 μl sample (cell media) or calibrator (synthetic peptide standards diluted in DMEM or DMEM/F12). After overnight incubation at 4 °C with gentle shaking, plates were washed 5 times for 10 min with PBS-T washing buffer. For Aβ40 ELISA, 100 μl Mono-HRP-conjugated-streptavidin (Pierce) (0.1 μg/ml) in Mono-HRP buffer (11 mM NaH₂PO₄, 85 mM Na₂HPO₄, 137 mM NaCl, (pH 7.4), 0.05% Tween-20, 6% PEG) or for Aβ34 and Aβ42 ELISA (for higher sensitivity), 100 μl Poly-HRP-conjugated-streptavidin (Pierce) (1:20,000 dilution) in Poly-HRP buffer (1.1 mM NaH₂PO₄, 8.5 mM Na₂HPO₄, 13.7 mM NaCl, (pH 7.4), 0.1% Tween-20, 5% BSA)) was added to the wells. Plates were incubated for 1 h at room temperature with gentle shaking and washed 5 times for 10 min with PBS-T washing buffer. For the initiation of enzymatic reaction, 100 μl 1-Step™ Ultra TMB-ELISA Substrate (Thermo Fisher Scientific Inc.) solution was added to the wells and the plates were incubated at room temperature in the dark for up to 30 min. To stop the reaction, 50 μl 1 M H₂SO₄, per well, was added. Using Synergy H1, BioTek Instruments Inc. plate reader, absorbance at 450 nm and 630 nm as a reference was measured. The data analysis was performed with Gen5 BioTek®software. For the fitting of standard curves obtained from the absorbance of calibrators, a non-linear four-parameter logistic fit without weighting was used as follows $$y=b_2+\frac{b_1-b_2}{1+{(\frac{x}{b_3})}^{b_4}}$$ where y is signal, x is concentration, b₂ is estimated response at the infinite concentration, b₁ is estimated response at zero concentration, b₃ is mid-range concentration and b₄ is slope factor.

Vaccination campaigns were mostly conducted through door-to-door visits. Central-point vaccination was performed only in El alia and Mornag municipalities, according to veterinary team's preparedness.
Vaccines were stored using classical cool boxes and refrigerated cooling devices with ice packs. Temperature inside these different devices was monitored using a thermo-button that recorded the temperature every 15 min. Thermotrack software (ProgesPlus) was used to collect data. Weather reports provided environmental temperatures. The veterinary staff was given a questionnaire addressing various aspects of cold chain and vaccination campaign logistics (Additional file 3).
Nobivac Rabies (MSD animal health), an inactivated rabies vaccine comprising at least 2 IU of Rabies virus strain Pasteur RV per dosage, 0.15 ml/ml of aluminum phosphate, and 0.01 percent thiomersal was used. Before distribution to vets, each vaccination batch was tested under National Drug Control Laboratory's procedures. Each dog received 1 ml dosage subcutaneously using dosing injection guns (Hauptner-Herberholz) made of stainless steel with a capacity of 30 ml and adjustable dosing with reusable 13G metal needles.