The FLOTAC dual technique was performed the next morning before new samples arrived. We used flotation solution 2 (FS2; selleck kinase inhibitor saturated sodium chloride [NaCl] solution; specific gravity [s.g.] = 1.20) and FS7 (zinc sulfate [ZnSO4?7H2O] solution; s.g. = 1.35). For the DNA isolation, we followed the procedure described by Verweij and others.39 All DNA samples were stored at ?20��C and transferred on ice to the NIMR-MMRC, where PCR amplification and detection were conducted in June and November of 2012. A multiplex real-time PCR was used for the simultaneous detection of A. lumbricoides, N. americanus, S. mansoni, and S. stercoralis DNA in fecal samples.31,32,40,41 For DNA amplification, 5 ��L DNA extracted from 0.1 g stool specimens were used as a template in a final volume of 25 ��L with PCR buffer (HotstarTaq Master Mix [5 mM MgCl2 and 2.

5 ��g bovine serum albumin]; Roche Diagnostics, Almere, The Netherlands), 2 pmol each A. lumbricoides-specific primer (Thermo Fisher, Ulm, Germany), 5 pmol each N. americanus-specific primer (Thermo Fisher), 5 pmol each Schistosoma-specific primer (Thermo Fisher), 2.5 pmol each S. stercoralis-specific primer (Thermo Fisher), 1.25 pmol each N. americanus-specific double-labeled probe (Biolegio, Nijmegen, The Netherlands), A. lumbricoides-specific double-labeled probe (Thermo Fisher), S. stercoralis-specific double-labeled probe (Biolegio), and Schistosoma-specific double-labeled probe (Thermo Fisher). Amplification consisted of 15 minutes at 95��C followed by 50 cycles of 15 seconds at 95��C, 30 seconds at 60��C, and 30 seconds at 72��C.

Amplification, detection and data analysis were performed with the Corbett Rotor-Gene 6000 Real-Time PCR System (Corbett Research, Mortlake, New South Wales, Australia) and Corbett Rotor-Gene 6000 Application Software, version 1.7.87 (Corbett Life Science, Cambridge, UK). Negative and positive external control samples were included in each amplification run. The details of all primers and detection probes used in our study are described elsewhere.31,32,40,41 Data management and statistical analysis. The helminth species-specific results derived by each method were entered manually in the participant’s CRF and subsequently transferred into a Microsoft Access 2010 electronic database (Microsoft Corporation 2010, Redmond, WA). Data were analyzed using STATA, version 12 (StataCorp., College Station, TX) and R, version 2.15.2 (R Foundation for Statistical Computing, Vienna, Austria).42 For the comparison of diagnostic methods, the diagnostic results of the first stool sample collected and examined from Batimastat each participant were included in the analysis.