**1. Introduction**

There has been a growing interest in understanding the composition of the human gu<sup>t</sup> microbiome and its linkages with all areas of health [1]. This has led to increased microbial community analysis, using techniques including 16 s rRNA gene sequencing, quantitative PCR, and culture-based methods [2,3]. Increasingly, large-scale observational and intervention studies have aimed to collect stool specimens to provide data in this rapidly evolving field of microbiology.

In both clinical practice and research studies, it has become increasingly common for stool specimen collection to be completed at an individual's home and then shipped to the laboratory [4,5]. At-home stool collection kits are often designed for a modern toilet seat and depend on a reliable national priority mail delivery system, [5,6] such as the widely used OMNIgene•GUT (DNA Genotek, Ottawa, ON, Canada) along with the OM-AC1 toilet accessory, a flushable collection paper secured to the toilet seat with adhesive strips [7].

Nevertheless, there is limited data concerning practical methods for collecting stool specimens for microbiome analyses in settings outside of the high-resource contexts, specifically for those without modern toilet seats or reliable shipment options. Thus, in an effort to characterize the human microbiome across the full range of the human experience, populations in low-resource settings continue to be underrepresented [8]. In the context of large studies in low-resource settings, specimen transportation and refrigeration, contamination, and acceptable collection may pose challenges. Designing a simple, user-friendly stool specimen collection kit for use in these challenging environments is imperative.

**Citation:** Fischer, J.A.J.; Karakochuk, C.D. Feasibility of an At-Home Adult Stool Specimen Collection Method in Rural Cambodia. *Int. J. Environ. Res. Public Health* **2021**, *18*, 12430. https:// doi.org/10.3390/ijerph182312430

Academic Editors: Diana María Cardona Mena and Pablo Roman

Received: 6 November 2021 Accepted: 25 November 2021 Published: 26 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Here, we report on study tools developed and used to collect neat stool specimens from women in rural Cambodia, used as a part of a randomized controlled trial that aimed to evaluate the potential harms of iron supplementation [9]. We discuss the feasibility and acceptability of our convenient at-home stool collection methods that have the potential to be implemented in similar low-resource locations.

#### **2. Materials and Methods**

#### *2.1. Study Participants and Setting*

This stool specimen collection methodology was designed as a part of a randomized controlled trial in rural Kampong Thom province, Cambodia, with ethics granted from the Clinical Research Ethics Board at the University of British Columbia in Vancouver, Canada (H18-02610), and the National Ethics Committee for Health Research in Phnom Penh, Cambodia (273-NECHR). All participants provided written informed consent before participating in the study, including the collection of baseline and 12-week venous blood, neat stool, and fecal swab specimen. Full details of the original study can be found elsewhere [9], and the trial was registered at clinicaltrials.gov (NCT04017598). Between December 2019 and January 2020, women were recruited, and *n* = 480 non-pregnan<sup>t</sup> women of reproductive age (18–45 years) were randomized to 12 weeks of daily oral iron supplementation in the form of either 60 mg ferrous sulfate (*n* = 161), 18 mg ferrous bisglycinate (*n* = 158), or placebo (*n* = 161). Venous blood specimens and neat stool specimens were collected at baseline and after 12 weeks.

Enrolled women did not have access to modern toilets with toilet seats nor household refrigeration. The most common type of sanitation facility available at most households was pour-flush to a septic tank or pit latrines (91% [437/480]).

#### *2.2. Stool Collection Protocol*

Development of the stool collection methodology took place in discussion with local public health staff highly experienced in rural specimen collection and knowledgeable about resource limitations of the study location (i.e., toilet facilities). Research staff were trained on the use of the stool collection kits and the procedures to disperse and collect the specimens from research participants. At the initial study visit, following administration of the baseline questionnaire, study staff provided the participants with the stool collection kit, verbally explained how to properly collect the specimen in Khmer (local Cambodian language), and dispose of collection materials. Women were also provided with a written copy of the same instructions regarding stool collection to take home via a simple Khmertranslated infographic. They were instructed to bring their stool specimen back to the local health centre the following day.

The stool collection kit was labelled with the participant ID number. It contained the following items: 30 mL clear polystyrene stool collection container with a screw-on blue lid and attached spoon, gloves, Khmer translated infographics and a metal pot (Figure 1). The metal pots were stored in heavy-duty plastic bags to prevent contamination before distribution to participants.

The verbal instructions as provided in staff training and written infographic (Figure 2) were communicated to study participants as follows:


**Figure 1.** Stool Collection Kit: (**a**) Resealable participant labelled bag, infographic, gloves, 30 mL clear polystyrene stool collection container; (**b**) Metal collection pot.

Women collected their stool specimens at their home and brought the completed kit back to the health center within ~2 h of defecation in the provided clear resealable plastic bag. Many participants opted to place the transparent bag inside a small opaque black garbage bag for additional privacy. Upon retrieval, study staff would ensure the stool collection kit contained the neat stool specimen and the container was tightly sealed. Tubes were labelled with the participant, study visit number, date, and time received. Labelled tubes were double-checked to ensure participant ID matched the ID number marked on the outer side of the bag. The kits were then immediately placed on ice. Neat stool specimens were transported on ice to the National Public Health Laboratory, where specimens underwent further processing and were frozen at −20 ◦C within 4–6 h until further analysis. Additionally, women were given the metal pot to keep and use for additional study visits where follow-up stool collection was needed.

Missing stool specimens were documented, and women were followed up by staff on the morning of the initial study visit. If a woman could not pass stool or was not available for a study visit that day, stool specimens were collected within seven days of the original study visit date. In this event, study staff called women to arrange another stool pickup, ensuring that pickup happened within 2 h of bowel movement and placed on ice, driven to the National laboratory and frozen at −20 ◦C within 4–6 h.
