Conventional Baermann (CB)

Ten grams of stool sample were weighed and mixed with two grams of activated charcoal and lukewarm water. The stool sample was transferred to a petri dish with a double layer of tissue paper at the bottom and then covered by single layer of tissue paper at the top to form a small pouch. Incubation was maintained for 18–24 hrs at 26°C. After incubation, stool samples were suspended for 1 hour in lukewarm water at room temperature (which is in our case between 25 and 37°C) and filtered using a conventional Baermann apparatus (a strainer on top of a funnel connected to a rubber hose clamped with a hemostatic clamp) supported by a funnel stand, the single layer tissue paper side of the pouch was facing the strainer, (Fig 2). Afterwards, the lower 10 mL from the water contained in the hose was drained off, centrifuged at 2000 rpm for 5 minutes and 1 mL of the sediment was examined microscopically for the presence of larvae [6]. The detail procedure is given in S1 Text.

Modified Baermann (MB)

Three grams of fresh stool sample was weighed and placed on cotton-wool gauze (8 layers–non-sterile) of 5x5 cm. A stool pouch was formed and placed on top of 50 mL falcon tube filled with lukewarm water just slightly touching the water surface (Fig 3). The tube was left to stand at room temperature (which is in our case between 25 and 37°C) for 2.30 hours. The supernatant was discarded and the 3 mL sediment was allowed to settle for 30 minutes and then examined under the microscope for the presence of larvae [4]. The detail procedure is given in S2 Text.

Modified Baermann with charcoal pre-incubation (MBCI)

This is a newly modified version of MB technique. The materials and procedures used were the same as MB but with addition of charcoal pre-incubation. Briefly, three grams of fresh stool sample were weighed and mixed with one gram of activated charcoal and lukewarm water. Then a stool pouch, as in the regular MB, was formed using cotton-wool gauze (8 layers–non-sterile) of 5x5 cm and placed in the center of a petri dish. Incubation was done for 18–24 hrs at 26°C, and then processed as in the regular MB. The detail procedure is given in S3 Text.

Reference standard test

Since there is no ‘gold reference standard’ for the Baermann technique, the results of all the three Baermann procedures were used in combination as a composite reference standard by which the presence of S. stercoralis larvae in at least one of the three Baermann procedures was used as a positive composite reference standard and the absence of S. stercoralis larvae by all the three Baermann procedures was used as a negative composite reference standard.