Enablers and barriers to stool sample collection for TB diagnosis: insights from caregivers and adult patients in three high TB/HIV burden countries in Sub-Saharan Africa (Eswatini, Mozambique, and Uganda)

Abstract

New stool-based TB diagnostics, such as Xpert Ultra, have shown promise in improving TB diagnosis in children and people living with HIV. However, limited research has explored the acceptability of stool collection among these target groups. This study aimed to assess the barriers and enablers influencing the willingness of caregivers of minor patients and adults to provide stool samples for TB diagnosis. We employed a generic qualitative study design that integrated different data collection approaches, including exit-, in-depth, and semi-structured interviews. This study was conducted in Eswatini, Mozambique, and Uganda between February 2022 and October 2024. A total of 102 participants, comprising 79 caregivers and 23 adult living with HIV, were recruited. Most participants reported no prior experience with stool sample collection. Nonetheless, they reported minimal difficulties in collecting stool samples and expressed a high willingness to provide stool samples. Factors influencing this willingness included the desire to recover from illness, trust in the healthcare system, and the non-invasive nature of the procedure. Refusals were rare, reported in 2 out of 102 participants. These were mainly attributed to challenges in obtaining stool samples at the required time and discomfort with handling the samples. Additionally, factors such as transportation costs to the health facility, direct association between sputum and cough, local beliefs surrounding stool were identified as potential barriers that may reduce willingness and shape patients’ preference for sputum-based methods over stool-based approaches, even among those otherwise open to providing stool samples. Our findings revealed that, despite high willingness among our participants to provide stool samples, several barriers could influence the acceptability of Sb-TBD and preference for alternative TB diagnostic procedures. These challenges should be addressed through strategies such as providing clear instructions for stool sample collection with collection kits, alongside community-based interventions to improve awareness and understanding of Sb-TBD.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12879-026-14154-0.

Clinical trial number

This study is part of the Stool4TB study, registered on 2021-09-08 under ID NCT05047315 on https://www.clinicaltrials.gov/.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12879-026-14154-0.

Background

Tuberculosis (TB) remains one of the world’s most significant public health challenges. According to the 2024 Global TB Report, an estimated 10.7 million people developed TB that year, with 11% being children and young adolescents [1]. The majority of TB cases occurred in the 30 high-burden TB countries, including Mozambique and Uganda, which collectively accounted for 87% of all global cases. Globally, 8.3 million new diagnosed cases were reported, and 1.23 million deaths were attributed to TB [1].

Evidence suggests that delayed detection remains the main challenge, largely driven by suboptimal health-seeking behaviors and limited diagnostic capacity [2–4]. Delayed diagnosis not only impacts on the microbiological cure from TB but also contributes to the burden of post-TB lung disease [5]. A central obstacle lies in the difficulty of obtaining good quality of sputum samples, particularly from young children and people living with HIV (PLWH), who often struggle to expectorate spontaneously [6–8]. In these immunodeficient patient groups, a reduced inflammatory response may inhibit sputum production [6]. Induced sputum techniques, nasopharyngeal aspirates, fiber-optic bronchoscopy, or the string test may all be used to retrieve pulmonary secretions from patients unable to provide a sputum sample, however, they present large logistical, cost, or biosafety challenges [6]. Moreover, obtaining other recommended specimens for confirming pulmonary TB in children requires invasive procedures (such as gastric aspiration or sputum induction), skilled clinical staff, and specialized consumables that are often unavailable in resource-limited settings [9, 10].

Beyond challenges related to the sputum sample, the global TB response continues to face major challenges, particularly because of its strong TB link with the HIV/AIDS epidemic, thus the need for innovative, affordable diagnostic tools [11]. To address this, the World Health Organization (WHO) has recommended stool testing using Xpert and Ultra for childhood pulmonary TB diagnosis [12–14]. These recommendations are informed by a large body of evidence showing that the use of other (biological) samples than sputum, such as stool-based testing, also enhances TB detection among PLWH, especially those with CD4 counts below 200 cells/µL, and could have additional diagnostic value to sputum- and urine-based tests [15]. Overall, these findings underscore the potential of stool-based procedures to enhance TB case detection among children and PLWH [16].

The Stool4TB collaboration is an EDCTP-funded project with 7 partners that evaluates a novel stool-based qPCR TB diagnostic assay in children and PLWH [2]. If the assay demonstrates good performance compared to a composite reference standard, including sputum (induced sputum and/or gastric aspirate in children) and stool Xpert Ultra, sputum culture (induced sputum and/or gastric aspirate in children), and urine LF-LAM (among PLWH) [17], and if specimen collection proves feasible and acceptable for both patients and healthcare workers, the test could have strong potential for national-level implementation [2].

While these findings provide useful insights into factors that may influence the acceptability of the stool-based TB diagnostics (Sb-TBD), there remains limited qualitative research specifically examining the patients’ and caregivers’ willingness or acceptance of this specimen collection method among its intended target populations, children and PLWH, particularly in the context of high TB/HIV burden countries [18].

This study aimed to explore barriers and facilitators influencing willingness to provide stool samples for TB diagnosis among caregivers of children and adults living with HIV in Eswatini, Mozambique, and Uganda.

Methods

Study design and setting

We adopted a generic qualitative research design - also referred to as basic qualitative or interpretive [19] - combining qualitative methods such as exit interviews and in-depth interviews. A generic qualitative design is appropriate when the aim is to understand participants’ experiences and their unique perspectives on those experiences. This approach is particularly useful for exploring individuals’ beliefs and opinions about their own realities and experiences, and those of others [20].

This study was conducted in Eswatini (Mbabane), Mozambique (Manhiça District), and Uganda (Kampala), three African countries with a high TB burden.

In Mozambique, the study was implemented by the Manhiça Health Research Center (Centro de Investigação em Saude da Manhiça, CISM), located in Manhiça, Maputo Province, in Southern Mozambique [21]. Manhiça is a rural district in Southern Mozambique, covering an area of 2360 Km2, with an estimated population of approximately 188,405 [2]. Most residents live in rural communities and rely primarily on subsistence farming and fishing, while others are employed as laborers in sugar cane plantations, sugar refining companies, or small agriculture companies [22]. The main local languages spoken are XiChangana and Xironga, and the dominant religions are Christianity and Islam. Southern Mozambique is a region that has reported very high rates of TB and HIV, both in the adult and pediatric population [23–25]. Similar to other countries in southern Africa, Mozambique is among the 10 countries with a high burden of tuberculosis (TB), TB/HIV co-infection, and multidrug-resistant TB (MDR-TB). The country currently has an estimated TB incidence rate of 361 cases per 100,000 population [26]. A study conducted at Maputo Central Hospital performed diagnostic autopsies on 223 deaths (56.5% HIV-positive), including children, maternal deaths, and other adults [24]. This study found that 62 cases (27.8%) had active TB disease at the time of death, while 80 cases (35.9%) showed pathological findings consistent with tuberculosis [24].

In Uganda, the study was conducted at the Mulago National Referral Hospital (MNRH), which is the principal public tertiary healthcare facility in Uganda, situated on Mulago Hill in the Kawempe Division of Kampala City. MNRH functions as the primary teaching hospital for the Makerere University College of Health Sciences, the oldest medical school in the region. Kampala, covering approximately 189 square kilometres, is the nation’s rapidly expanding urban area, with an estimated population of 2,503,174 [27]. The informal sector dominates the city’s socio-economic landscape, featuring densely populated and low-resource areas with major urban slums, including Katanga, Kisenyi, and Bwaise. The predominant language spoken is Luganda, although English and Swahili are the official languages. As a national referral centre, the MNRH receives complex cases, including tuberculosis (TB), from lower-level facilities. The public health challenge is compounded by high infectious disease prevalence; specifically, the central region and Kampala have an adult HIV prevalence of up to 8.1%, higher than the national average [28].

In Eswatini, the study was conducted across three government-owned health facilities (Piggs Peak, Dvokolwako, and Mbabane), all in the Hhohho region, and two non-governmental facilities (Baylor Children’s Foundation Center’s of Excellence in Manzini and Mbabane). The data reported here were obtained from the Dvokolwako Health Center and Pigg’s Peak Government Hospital, which serves as the regional referral hospital. The Hhoho region lies in the north-western part of Eswatini, covering an area of 3625.17km2, and had a population of 320,651 [29]. The region is home to 26 government health facilities, including two hospitals, two health centers, and 22 clinics, excluding privately owned facilities.

Participants and sampling

The study participants included caregivers of children under 8 years and adults living with HIV presenting with TB symptoms. Before their enrollment in the clinical study and then consultation, caregivers and adults with HIV were introduced to the qualitative study and were informed about the overall Stool4TB project by a trained clinician. Following their consultation, a social research assistant provided a more detailed explanation of the study and invited participants to participate in the study by signing an informed consent form. We employed a combination of purposive sampling with convenience sampling [30] to select participants according to predefined criteria: being 18 years or older, responsible for a child aged under 8 years or living with HIV as a presumptive TB case, having sought care at the main study health facility, being enrolled in the stool4TB clinical study, and providing consent to participate in the socio-behavioural study (SBS). Participants were excluded if they were not the same caregivers who had consented to the main study, had temporarily relocated outside the study catchment area, or had a health condition that did not allow them to engage in an in-depth conversation. Eligible participants were approached after completing their clinical consultation by a research assistant specifically assigned to recruitment for the qualitative study. No eligible participants declined to participate across the three study countries.

In Mozambique and Uganda, data collection continued until data saturation was reached, defined as the point at which no new information, codes, or themes emerged from the data, and additional data collection no longer contributed to new findings (i.e., data became redundant) [31, 32]. In Eswatini, information power was used to determine the adequacy of the sample size, considering available resources and budget constraints. Data collection continued until sufficient information had been obtained to adequately address the research questions, guided by the five dimensions of information power: (ii) a specific and relatively homogeneous sample, comprising caregivers of children under 8 years of age and adults living with HIV presenting with TB symptoms; (iii) the application of a relevant theoretical framework, namely the socio-ecological model; (iv) the quality of dialogue during data collection, supported by strong researcher–participant interactions that facilitated in-depth discussions; and (v) the analytical strategy, which involved deductive thematic analysis guided by the study objectives and conceptual framework [32]. Nevertheless, we acknowledge that the relatively small sample size in Eswatini may have limited the opportunity to capture a broader range of perspectives, identify unanticipated issues that were not considered during the study design phase, and conduct more comprehensive cross-country comparisons.

Data collection and procedures

Data were collected between February 2022 and October 2024 at Mulago Hospital (Kampala, Uganda), Manhiça District Hospital (Manhiça, Mozambique), Pigg’s Peak Government Hospital, and Dvokolwako Health Centre in the Hhohho region (Eswatini).

Different qualitative data collection methods were adopted across the three study sites due to contextual and operational considerations. These differences were largely driven by budget constraints, particularly in Eswatini and Uganda, which affected the timely recruitment of social science leads, establishment of the qualitative research teams, and completion of the ethical approval process. Staff availability and the need to align the qualitative component with the progress of the clinical study also influenced the choice of data collection methods.

In Mozambique, the qualitative study commenced while the clinical study was still recruiting both caregivers of children and adults living with HIV, allowing the use of a combination of exit and in-depth interviews.

Exit interviews (EIs) were conducted using open-ended questions designed to explore the participants’ experiences and perspectives on providing a stool sample for TB diagnosis (Supplement 1). These interviews were conducted immediately after participants left the consultation room, within the health facility, at a location of their choice, following consent, and lasted between 20 and 45 min.

In addition, in-depth interviews (IDIs) were conducted to gain a deeper understanding of the previously recruited participants’ experiences with stool sample collection and their satisfaction with the procedure (Supplement 2). These took place after participants received their results, at a location of their choice based on comfort and preference, and lasted between 30 and 60 min.

In Eswatini, although the clinical study was also recruiting both participant groups, budget limitations restricted the scope of the qualitative component, influencing both the choice of semi-structured interviews and the achievable sample size. In Uganda, the qualitative component started later, after recruitment of adults living with HIV had already been completed, leaving only caregivers of children eligible for enrolment. Consequently, semi-structured interviews were adopted as the primary data collection method in both Eswatini and Uganda, reflecting the available resources and the stage of implementation of the clinical study.

In both countries, the semi-structured interviews examined participants’ perceptions of Sb-TBD and their experiences with the collection process (Supplement 3). Similar to the IDIs, these interviews were conducted after participants received their results, at a location of their preference, and lasted between 30 and 60 min.

In all three countries, the research team consisted of two research assistants fluent in both the official and local languages who were trained in the study protocol and qualitative data collection procedures. They were supervised by a site coordinator who reported directly to the Principal Investigator of the social science component of the Stool4TB project. Interviews were conducted in Changana and Portuguese in Mozambique, Siswati and English in Eswatini, and Luganda or English in Uganda, according to participants’ preferences. The interviews were digitally recorded with the participants’ consent. In instances where consent for recording was withheld, detailed field notes were recorded. All recordings and field notes were securely stored on the CISM, Makerere University, and Baylor’s server, ensuring that only study members had access to the data [22].

Data management and analysis

After data collection, field notes were summarized directly on the interview guide sheets, and all recordings were transcribed verbatim by research assistants. During transcription, the recordings were translated into English in Eswatini and Uganda and Portuguese in Mozambique. Selected illustrative quotes from Mozambique were translated from Portuguese to English for inclusion in this manuscript. To ensure confidentiality, all summaries and transcripts were anonymized using a unique participant code, starting with the type of participant, order of recruitment, and country of origin (e.g., Adult 11, Mozambique or Caregiver 11, Eswatini). During transcription, repeated words, incomplete sentences, and hesitation markers were removed for readability, while missing or implied words were added in brackets to preserve the meaning and ensure clarity [22].

To ensure data accuracy and consistency, the study team reviewed the recordings and transcripts throughout the study [22]. Initially, each site coordinator listened to every recording to verify that all questions were asked as outlined in the interview guide, assess response quality, and identify emerging themes. The site coordinator with the social sciences component Principal Investigator then reviewed these assessments to design corrective measures to improve data quality, which were discussed and implemented by the research assistants [22].

Quality control of the transcripts followed a similar process: each transcript was compared with its corresponding audio recording, and any discrepancies or omissions were documented and corrected accordingly. In cases of disagreement, the site coordinator, along with the consortium coordinator and principal investigator, discussed the issue until reaching a consensus. Once finalized, all corrected transcripts were archived for subsequent analyses [22].

These debriefing and quality assurance activities served as important reflexive processes and were maintained throughout data collection, analysis, and interpretation. Regular discussions among research assistants, study site coordinators, and the Principal Investigator, as described above, facilitated critical reflection on how researchers’ professional backgrounds, perspectives, assumptions, and prior experiences could influence data collection, coding, and interpretation. The research team also reflected on potential power dynamics between interviewers and participants and implemented strategies to promote a respectful and supportive environment where participants felt comfortable and empowered to express their views freely.

The data were analyzed using a combination of inductive and deductive thematic analysis approaches, which provided a structured yet flexible framework for identifying, analyzing, and interpreting patterns of meaning within datasets [33]. During data collection, an inductive approach was adopted to allow participants’ experiences with SbPs, and perspectives to emerge from data without being constrained by predefined categories. This inductive approach informed the development of the initial codes and themes, including participants’ prior experience with stool-based procedures (SbPs) and challenges encountered during stool sample collection [22]. Within each topic, specific code and sub-code were developed to capture relevant patterns in the data (Table 1). For example, codes were used to identify the presence or absence of specific experiences or perceptions, while sub-codes captured more detailed aspects of participants’ responses (positive or negative experience).

Table 1.

Code mapping

Theme	Sub-theme & Codes	Description	Summary of findings	Illustrative quote
Experiences with stool-based procedures	Previous experience providing stool samples	Participants’ previous exposure to stool sample collection and their understanding of the purpose of stool-based examinations	Low or no prior experience in providing stool	“For me it was a challenge at first. I eventually accepted it because I’ve had to take the stool a couple of times” (Adult 01, Eswatini). “I never offered it, but I had a nephew who was asked for his faeces” (Caregiver 36, Mozambique).
	Positive experiences with stool sample collection	Experiences characterized by comfort, acceptance, or perceived ease of providing stool samples	Perceived ease of sample collection	“It was easy. The strategy was to put the faeces in a jar, which had a small spoon inside, and I took a little out with that spoon. I kept it well, when I put it in the jar, until there was no dirt left, I kept it well, cleaned it well and then closed it. I put it inside the plastic bag […]” (Adult 09, Mozambique). “I found it easy, […] because my child wears a diaper. When I saw that they had pooped, I scooped some into the container and kept it in a cool place. The difference is that I didn’t bring it to the hospital but Baylor came to collect it the following day. I wrapped the container with toilet paper. I then placed the container in the plastic bag provided and sealed it” (Adult 09, Eswatini).
	Negative experiences with stool sample collection	Experiences associated with discomfort, embarrassment, difficulty, or concerns related to stool handling	Discomfort and challenges in producing and handling a stool sample	“The difficult part was the faeces. It was difficult because of the way it had to be carried, […] since it’s dirty […]. Yes, to help me with how to do it, they told me to find a leaf to use to put it in. I covered it, put it [in the jar], then covered it, put it in the plastic bag and washed my hands” (Adult 10, Mozambique). “At first, it was a bit difficult. I told the child to defecate, but it was difficult for him. The feces had refused to come out because, during that time, he was not eating well. I also wondered how I would move with feces” (Caregiver 03, Uganda).
Willingness to provide stool samples	Facilitators to providing stool samples	Factors that encouraged willingness to provide stool samples, categorized according to socio-ecological levels (Individual, interpersonal, organizational, community, and societal factors)
	Intrapersonal factors	Individual-level factors, including knowledge, previous experiences, attitudes, perceived benefits, and beliefs regarding stool-based TB diagnosis	Challenges producing sputum	“Yes, I will deliver it. There are things that can be difficult, […] specifically […] sputum, because […] it doesn’t come out, nor does the saliva from the sputum come out. I just cough air. That’s what will be difficult” (Adult 12, Mozambique).
	Interpersonal factors	Interpersonal factors, comprises the participant’s social network including family, friends, peers, and health care providers;	Peer and family experiences with SbPs	“I was not surprised because I have seen certain children or mothers commenting on or bringing those tiny little bottles or jars for urine and feces” (Caregiver 11, Mozambique). “I never offered it, but I had a nephew who was asked for his faeces” (Caregiver 36, Mozambique).
	Organizational factors	Institutional-level factors, including formal and informal rules and regulations operating within health facilities.	Perceived need to comply with institutional procedures	“I’ll hand it [stool] over, as is hospital policy. They want to treat me, but it’s difficult” (Adult 10, Mozambique). “I can’t refuse. If I refuse my health will get worse. This method of diagnosing is the one we will have to live with. I don’t see how you can change when it has been approved. […] they searched until they found it and discovered it was hiding in the stool. They want us to be healthy” (Adult 03, Eswatini).
	Barriers to providing stool samples	Factors discouraging willingness to provide stool samples, categorized according to socio-ecological levels (Individual, interpersonal, organizational, community, and societal factors shaping preferences for different diagnostic approaches)
	Intrapersonal factors	Individual-level factors, including knowledge, previous experiences, attitudes, perceived benefits, and beliefs regarding stool-based TB diagnosis	A sense of aversion to stools and difficulty providing a stool sample on demand	“I don’t have the courage to show it, [rather] I prefer to give sputum, which I can cough up right there and offer” (Adult 07. Mozambique). “I would refuse because it is […] difficult to get stool from the child because sometimes you ask for it but the child does not want to pass” (Caregiver 19, Uganda).
	Interpersonal factors	Interpersonal factors, comprises the participant’s social network including family, friends, peers, and health care providers.	Cumulative household health burden	“What could change is that I would have to look for transport to go to the hospital. I already have a child with sickle cell disease, and it takes a lot to care for them. When I add a child with TB, it becomes a lot. Sometimes you might fail to get the money to take them to hospital” (Caregiver 01, Uganda).
	Institutional factors	Institutional-level factors, including formal and informal rules and regulations operating within health facilities.	Limited understanding of the purpose of the examination and how to collect the stool sample	“I thought maybe they wanted to see if there was another infection in the stool disturbing him other than TB. Therefore, I thought that if they did not discover TB, they would find another disease through the stool that caused him to have a persistent cough. Yes, that is what I believe. I thought for TB, they were to use sputum” (Caregiver 01, Uganda). “[…] it was difficult. [sigh] It is not easy, but [sigh] the hardest part […] was collecting the poop [stool]. When they gave me the container, I got home in the morning. […] she (the children) went in a basin, and then I transferred it to the jars, even with some pee [urine]. I put it in the jar and then wrapped it in plastic” (Caregiver 26, Mozambique).
Preferences for TB diagnostic procedures	Preference for stool-based TB diagnosis	Participants expressing preference for Sb-TBD	Preference for stool-based procedures due to ease of sample collection among individuals unable to produce sputum	“With children, I can only bring their faeces, because as they are still children, they cannot cough and spit it out. They cough and swallow it” (Caregiver 06. Mozambique). “I prefer […] the poop one because it is easy. Whenever the child would tell me that they want to […] poop I would tell him to put it in the container” (Caregiver 14, Uganda).
	Preference for alternative diagnostic procedures and factors influencing diagnostic preferences	Participants preferring other diagnostic methods and reasons underlying this preference (Individual, interpersonal, institutional, community, and societal factors)	Preference for sputum-based procedures due to their perceived direct association with TB-related cough, lower unpleasantness than stool, and ability to be produced on demand	“I prefer sputum. Hiii (expressed a feeling of disgust), picking up feces, nothing. Eish, even picking it up today took courage, it’s not pleasant […]” (Caregiver 35, Mozambique). “[I would prefer] the sputum, because it is a direct method” (Caregiver 15, Uganda). “I prefer to give a sputum sample, which I can cough up right there and offer, whereas poo is difficult” (Adult 07, Mozambique).

Subsequently, a deductive approach was applied to guide the interpretation and organization of themes by integrating the predefined research themes (acceptability and preferences) with constructs from the socio-ecological model as the analytical framework. This enabled the research team to examine how factors influencing the acceptability of Sb-TBD operated across different levels, including intrapersonal or individual, interpersonal, community, institutional and societal factors [34].

The constructs of the social-ecological framework were applied as a code to identify and analyze the key factors emerging from participants’ accounts that influenced participants’ willingness to provide stool samples and their procedural preferences. These included intrapersonal factors, referring to individual attributes such as knowledge, experience, attitudes, behaviors, self-concept, and skills; Interpersonal factors, comprising the participant’s social network including family, friends, peers, and health care providers; institutional factors, encompassing formal and informal rules and regulations operating within health facilities; community factors, which include relationships among organizations and institutions, informal networks, and prevailing cultural norms within defined settings; and societal factors, referring to broader national laws, policies, and structural influences [34, 35]. These constructs were used to categorize factors acting as barriers or facilitators across different levels of influence, helping to explain the reasons underlying favorable or unfavorable perceptions and procedural preferences.

Three research team members were involved in the coding process, including two research assistants and the site coordinators. Data were organized and coded using a structured Microsoft^® Excel^® 2010 matrix, with participants’ IDs in the rows and themes, sub-themes and codes, and summary of findings in the columns, allowing mapping of responses at the intersection of participants and analytical categories. The coding process was conducted collaboratively, with consistency amongst coders discussed and refined through iterative review meetings, as previously described. Sub-theme summaries were then developed by synthesizing coded data, highlighting key patterns and divergences across participants. These summaries were supported by illustrative direct quotations selected to reflect both similarities and differences across participants. The analysis progressed from descriptive organization of coded data to interpretive synthesis, resulting in thematic narratives supported by evidence from the dataset. Where appropriate, findings were triangulated with existing literature to enhance the validity of the results. Reporting of findings followed the Consolidated Criteria for Reporting Qualitative Research (COREQ) guidelines [22].

Results

Sociodemographic characteristics

In this study, we interviewed 79 caregivers of pediatric patients (40 from Mozambique, 36 from Uganda, and 3 from Eswatini) and 23 adult living with HIV (20 from Mozambique and 3 from Eswatini). Overall, most participants were younger and female adults, with adult patients generally older than caregivers. Differences were also observed between the two groups, as caregivers were more likely to be married and have higher levels of literacy than adult living with HIV (Table 2).

Table 2.

Sociodemographic characteristics

Characteristics	Adult living with HIV Total (n = 23)	Caregivers Total (n = 79)
Age Group
Young adults (18–35)	5 (21.7%)	56 (70.9%)
Adults (36–65)	15 (65.2%)	23 (29.1%)
Elderly (66+)	3 (13.0%)	0 (0.0%)
Gender
Female	14 (60.9%)	74 (93.7%)
Male	9 (39.1%)	5 (6.3%)
Marital status
Single	14 (60.9%)	27 (34.2%)
Married	7 (30.4%)	46 (58.2%)
Widowed	2 (8.7%)	6 (7.6%)
Education Level
Illiterate	10 (43.5%)	4 (5.1%)
Incomplete Primary level	6 (26.1%)	15 (18.9%)
Primary level completed	3 (13%)	6 (7.6%)
Incomplete Secondary level	3 (13%)	11 (13.9%)
Secondary level completed	1 (4.3%)	27 (34.2%)
Tertiary education	0 (0.0%)	16 (20.3%)

Caregivers’ and adults’ living with HIV experiences in providing stool samples for diagnosis of diseases

Our findings from Mozambique and Eswatini indicated that, among the respondents who provided information on previous experiences with SbPs, approximately 27 of 32 reported that providing stool samples for TB diagnosis was a first experience, as stated by the following respondent: “It was my first time” (Adult patient 05, Eswatini). The remaining five (15.6%) respondents, mainly from Mozambique, reported previous exposure to the SbPs, either through their own experience or through experiences shared by family member or peers.

I never did, but I have a nephew who was asked for feces (Caregiver 36, Mozambique).

Caregivers’ and adults’ perspectives on stool sample collection for TB diagnosis

Regarding the sample collection experience, nearly 21 of the 25 respondents reported having had a positive experience, emphasizing that the stool was from someone familiar and that the collection posed no difficulties. Some caregivers noted that their children had no trouble defecating and they were able to collect the samples themselves. Some described using practical strategies, such as defecating or asking their children to do so, on the ground, on a plastic sheet, or on another surface, and then collecting the sample with a wooden stick. Several participants shared their experiences, explaining the following:

It was not difficult because I was dealing with my own child. It is not a neighbor’s child or someone else’s child but mine, all I want is his health. After the health it doesn’t matter what I got (Caregiver 4, Uganda).

Reinforcing this perspective, another participant stated:

It is a short [to collect stool sample]. It was easy because the end result was to determine what was wrong with my child. I found it easy. Having a sick child is stressful, which is why I decided to find out what was wrong with the child. I can do anything to find out what is wrong with my child and how they can be treated. I find it to be an easy way to run the TB test because every child poops (Caregiver 1, Eswatini).

Only a few (4/25) participants reported difficulties in collecting stool samples, which, in some cases, stool sample were mixed with urine. These challenges were primarily related to discomfort in handling stool or challenges in getting the child to defecate.

For me, it was a challenge at first. I eventually accepted it because I had to take the stool a couple of times (Caregiver 3, Eswatini).

This experience was not isolated, as another participant from Uganda described:

At first, it was a bit difficult. I told the child to defecate, but it was difficult for him. I also wondered how I would move with feces. The feces had refused to come out because, during that time, he was not eating well (Caregiver 3, Uganda).

This was further supported by a caregiver from Mozambique:

[…] it was difficult. [sigh] It is not easy, but [sigh] the hardest part, the hardest part, was collecting the poop [stool]. Hmm. When they gave me the container, I got home in the morning. […] she (the children) went in a basin, and then I transferred it to the jars, even with some pee [urine], I put it in the jar and then wrapped it in plastic. Hmm [yes]. I brought it here. It was in plastic. It was in the bowl. Ah, […] I used plastic. It was not easy, but since they asked, and for her health. [So] I had to do it. Hmm [yes] (Caregiver 26, Mozambique).

Future willingness to provide stool sample: barriers and enablers

When assessed for hypothetical future willingness, around 92 of the 94 respondents across all three countries expressed future willingness to provide stool samples for future TB diagnosis. For instance, an adult patient living with HIV reported being as adherent to the instructions as possible with no embarrassment, and trust in local health services.

Yes, I would provide [stool samples if they ask me in the future]. I did what the doctors instructed. It was the same; I didn’t ignore anything and didn’t refuse anything. I didn’t feel embarrassed at all. I just gave thanks, because as long as I get the medication, this disease will go away (Adult patient 11, Mozambique).

Low post-experience acceptability was observed among two caregivers, from Uganda and Mozambique, who had previously undergone the stool-based qPCR test, expressed unwillingness to repeat the procedure in the future.

I would refuse because it (giving stool) is not good (Caregiver 19, Uganda).

Enablers of acceptability

From the perspective of the social-ecological model, only factors within the intrapersonal, interpersonal and institutional dimensions were identified as influencing the acceptability of caregivers and adult patients living with HIV. Among participants who expressed willingness, acceptance was primarily shaped by intrapersonal factors such as their desire to recover from illness, as well as by the perceived simplicity of the sample collection process, particularly when compared to sputum induction. As one adult patient, from Eswatini noted, “I cannot refuse. If I refuse, my health will get worse”. This perspective was echoed across respondent groups. The perceived ease of stool collection was especially emphasized by caregivers of minors who had previously experienced sputum induction procedures.

The only challenge I encountered as a parent was when I had to hold the child while being tested; it really scared me as a parent. The tubes they put in the nose were very scary. The collection of urine and fecal samples was easy. Yes, it (stool) was easy, but the other methods were not. In particular, sputum collection is not easy. […] It really affected me. I saw the child crying; it was really traumatizing (Caregiver 06, Uganda).

Additionally, institutional factors, including the perceived need to comply with local healthcare procedure and trust in the health institution, particularly in its expertise, technologies, and diagnostic procedures, together with interpersonal factor, such as testimonies from other people and peers who had undergone similar SbPs, further encouraged compliance with medical instructions.

I’ll hand it [stool] over, as is hospital policy. They want to treat me, but it’s difficult (Adult 10, Mozambique).

My trust, like I said before, I trust the health workers within the recognized setting because they know what they are doing. So, compared to me going to a private setting and people don’t have the equipment, they don’t have the reliable storage and probably the equipment to use. […] I have enough confidence in those centers that they have enough technology that they use to detect what they want to detect either TB or every illness (Caregiver13, Uganda).

[…] I have seen certain children or mothers commenting on or bringing those tiny little bottles or jars for urine and feces (Caregiver 11, Mozambique).

Barriers to the acceptability

With regard to barriers, we identified several factors within the intrapersonal dimension. In particular, the two participants who were unwilling to provide a stool sample attributed their reluctance primarily to the difficulty of producing a sample on demand and to their perception of the procedure as dirty and unpleasant.

I would refuse because it is not good. It is difficult to get stool from the child because sometimes you ask for it, but the child does not want to pass it (Caregiver 19, Uganda).

I find it strange (laughs) because bringing stool to the hospital is strange. For me, I would never [bring it] […] because stool is one of those dirty things that (laughs) no one can stand (Caregiver 06, Mozambique).

Interestingly, the view of stool as “dirty” was expressed across respondents in all countries, even among those willing to provide stool samples in the future. Additionally, some participants, particularly from Mozambique and Eswatini, associated stool collection with witchcraft, as illustrated by one caregiver from Eswatini:

I would give it to you because I do not know you. I would not give it to a person I know. We no longer trust others. We will not give it to people we know. We give it to you because we do not know you well. People do many bad things. Let me give you an example: people can put a (sicitfo) on your stools. There is much that can be done with stool samples. If there is a person that likes to go around leaving their stool or leaving their stool under the tree or on the road side. Others will put (sthombhomnandi -they say the anus comes out) I don’t know what happens or the anus will shrink and when they need to do their stools I don’t know what they do. People do different things. People do wicked things out there (Adult patient 05, Eswatini).

These findings indicated that additional institutional/structural factors, such as the distance to health facilities and unclear explanation of the purpose of the technique, beyond those reported by participants who refused, may influence patients’ willingness to provide stool samples. For instance, three participants mentioned the cost and logistical challenges of taking samples to the health facility. As one Ugandan caregiver (01) explained:

What could change is that I would have to look for transport to go to the hospital. I already have a child with sickle cell disease, and it takes a lot to care for them. When I add a child with TB, it becomes a lot. Sometimes you might fail to get the money to take them to hospital (Caregiver 01, Uganda).

This indicates that, at the interpersonal level, some participants described a TB diagnosis not as an isolated health event, but as a compounding burden that exacerbated existing household vulnerabilities. For instance, the above quote illustrates that families already struggling with other health issues [e.g., sickle cell disease], the requirement to manage a TB diagnostic process, even a non-invasive one like stool collection, was perceived as an overwhelming addition to their current hardships. This suggests that the acceptability of new diagnostic tools is heavily influenced by the pre-existing cumulative stress within the family unit.

Additionally, unclear explanation of the technique’s purpose may have contributed to misunderstandings about the rationale for sample collection and the inadvertent mixing of samples, consistent with the experiences reported by participants during stool sample collection. For instance, some participants believed that stool samples were being collected to test for other diseases, as illustrated by one Ugandan caregiver (01):

I thought maybe they wanted to see if there was another infection in the stool disturbing him other than TB. Therefore, I thought that if they did not discover TB, they would find another disease through the stool that caused him to have a persistent cough. Yes, that is what I believe. I thought for TB, they were to use sputum (Caregiver 01, Uganda).

Preference for Sb-TBD

Although most (92/94) respondents expressed a willingness to provide a stool sample for TB diagnosis, approximately 64 of them preferred sputum-based methods, including sputum induction, over stool-based approaches (24/92, especially in Mozambique and Uganda. Six respondents expressed not preference between both the two sample collection methods. Intrapersonal factors, such as the direct association of sputum with coughing and TB exam, the unpleasant smell of stool and the ability to be produced on demand, played a significant role in this preference. However, this tendency was observed mainly among caregivers of children, particularly those who did not have a negative experience with sputum induction.

[…] I would choose the tubes. They are not disgusting. Yeah, it is scary but not disgusting. No, according to me the sample I would recommend to be maintained is that one of the tubes. Yes, it should be maintained. Because, like I told you that TB is spread through the air and as soon as one gets it, it goes straight to the lungs and in the same lungs, they get the sputum which means TB can only be found in the lungs since they store the air that one inhales (Caregiver 8, Uganda).

I prefer to give a sputum sample, which I can cough up right there and offer, whereas poo is difficult. (Adult 07, Mozambique).

Discussion

In this study, we analyzed barriers and enablers to the acceptability of stool samples for TB diagnosis among caregivers of children < 8 years old and adults with HIV presenting with TB symptoms. Overall, our findings indicate that for most respondents, this was their first time both providing a stool sample and learning about Sb-TBD, and they reported no difficulties in collecting the sample. Among those who reported challenges, these were primarily associated with feelings of discomfort or disgust when handling stool. Contrary to our findings, a related study on stool-based testing for childhood tuberculosis reported that the main challenges were estimating the correct stool volume, collecting solid stool with a wooden stick, and laboratory difficulties, such as distinguishing between supernatant and sediment [18].

When describing the strategies used to collect stool samples, participants reported employing various practical methods, including defecating on a surface or on a plastic sheet, and then transferring the sample using a stick or plastic material to avoid contamination with urine, soil, detergent, or toilet disinfectants, in accordance with the Standard Operating Procedures (SOPs) provided to healthcare workers involved in the Stool4TB project. Nevertheless, one participant reported that stool sample had become mixed with urine during collection. Although urine contamination may have limited impact on molecular testing, this finding underscores the importance of giving patients and caregivers clear instructions on proper stool collection and ensuring they receive a complete and appropriate collection kit [36], rather than relying solely on verbal guidance as was done in the Stool4TB clinical study. Supporting this, a literature review on the role of stool testing in diagnosing and monitoring gastrointestinal disorders in children emphasized that stool samples should not come into contact with urine, water, toilet paper, or diapers [37]. Such contact can compromise the accuracy of results, as toilet papers often contain sugars (such as cellulose) and diapers absorb water, potentially altering the sample composition [37]. In this regard, two related studies on patient attitudes toward fecal sampling for gut microbiome research and clinical care found that many participants considered the collection process easier when an instruction sheet was included with the collection kit [38, 39].

Despite their limited knowledge of Sb-TBD and prior experience with SbPs, most participants expressed a strong willingness to provide their own or their child’s stool sample for TB diagnosis in the future. Consistent with this finding, previous studies assessing the acceptability of stool sampling for the diagnosis of gastrointestinal diseases reported generally high levels of acceptability [38, 40].

In our study, the high willingness to undergo stool-based testing was primarily driven by the participants’ desire to recover from illness, the perceived simplicity of the sample collection process, peers’ testimonies, and trust in the health institutions, including confidence in the knowledge and technology employed. Similarly, existing evidence from three studies demonstrates that participants’ attitudes toward fecal sampling were driven by their willingness to comply with the procedure and recover [2, 38, 40].

While this may suggest a largely voluntary willingness to comply, a similar study conducted in Manhiça District highlights the influence of power dynamics between the government and healthcare providers, as well as between healthcare providers and patients. Within this context, the study suggests that both patients’ willingness to comply with medical instructions and healthcare providers’ efforts to enforce compliance are driven not only by the desire to cure illness, as our findings indicate, but also by the imperative to adhere to national guidelines, if Sb-TBD were formally approved by the government [2]. Another study carried out in Mozambique illustrated how power dynamics between healthcare providers (midwives) and patients (women in labor) could manifest as (non-) physical violence by healthcare providers [41]. This behavior operated within what the authors describe as a “salvation discourse,” in which aggression is framed as an expression of care and concern for life, while simultaneously functioning as a means of exercising professional power and authority [41]. Together, these two studies suggest that patients’ seemingly voluntary or compliant adherence to Sb-TBD may not reflect genuine acceptance, but rather a psychological self-protection strategy aimed at minimizing tension with healthcare professionals and safeguarding continued access to healthcare services [42]. Although this was not directly explored in the present study, these findings may suggest that power dynamics and psychological self-protection could influence participants’ responses and willingness to comply with diagnostic procedures, particularly in contexts characterized by existing power asymmetries between healthcare systems, providers, and patients. These factors should therefore be considered in future research examining the implementation and acceptability of Sb-TBD.

The non-invasive nature of the procedure emerged as another key motivating factor, particularly among caregivers of children who had a traumatic experience with induction. This finding is consistent with existing evidence from Mozambique, which highlighted caregivers’ preference for Sb-TBD over sputum induction (or respiratory sampling) due to their non-invasive nature [2]. This finding reinforces the notion that patients’ willingness to adhere to the technique is primarily driven by their desire to recover or comply with medical recommendations, rather than by the relative advantages of the method, such as its non-invasive nature and the fact that it can be provided by anyone, when compared to standard sputum-based procedures [2].

Although refusals were rare and occurred mainly among female caregivers, they were driven by two factors, such as discomfort and feelings of disgust associated with handling stool samples and practical challenges, including the difficulty of producing a stool sample on demand. This contrasts with findings from another study that reported notable differences by gender and education level, with women being more likely than men to provide a stool sample and individuals with higher educational attainment being more willing to do so [38]. In contrast, a similar study conducted in Manhiça District (Mozambique), examining healthcare providers’ perspectives on the acceptability of Sb-TBD, suggested lower acceptability among literate individuals [2]. This was attributed to factors such as their type of employment, working hours, and higher health literacy. In our study, limited demographic data, particularly the underrepresentation of men, and the lack of national representativeness precluded analysis of the relationship between participants’ characteristics and their willingness to participate.

Our findings indicated that the perception of stool as unpleasant was the main cited reason participants refused and most preferred sputum-based procedures over Sb-TBD. These findings are consistent with two similar studies on stool sampling acceptability, which reported that participants who declined to provide samples claimed the unpleasant nature of stool collection and cultural taboos related to the perceived “dirtiness” of human feces as key reasons [39, 40]. Furthermore, a study conducted in Mozambique cautioned that the concept of Sb-TBD could generate confusion, skepticism, or even outright rejection, particularly because the link between stool and respiratory infection may seem counterintuitive, as well as between stool and witchcraft [2]. Consistent with this, our findings suggest that the direct association of sputum with TB contributes to participants’ preference for sputum-based procedures. In addition, local beliefs surrounding stool may further discourage patients from providing samples, particularly in low-income settings and in contexts where stool is strongly associated with witchcraft, as observed in Eswatini and Mozambique.

Refusal was also linked to practical challenges, including the difficulty of producing a stool sample on demand [2, 36]. Furthermore, the financial burden of transporting samples to the health facility may deter patients from returning the following day. Supporting this, a study conducted in Mozambique similarly found that barriers to acceptability would arise from intrapersonal factors, such as distance to health facilities, transportation cost, and limited prior experience with stool-based procedures [2]. Overall, this study found that although participants demonstrated high acceptability of Sb-TBD, many still preferred sputum-based diagnostics, highlighting the need for community-based interventions to increase awareness and understanding of Sb-TBD.

Strengths and limitations

To the best of our knowledge, this is the first study examining the willingness to provide stool samples for TB diagnosis among caregivers of children under 8 years old and adults living with HIV presenting with TB symptoms, from Mozambique, Eswatini, and Uganda. This study aims to help bridge the gap in the literature regarding the barriers and facilitators affecting the acceptability of Sb-TBD, particularly in countries with high TB burdens. However, several limitations should be acknowledged. First, the overrepresentation of female participants, particularly caregivers, which limited the ability to explore potential differences in perceptions and experiences between male and female participants. Second, due to logistical constraints, Uganda recruited only caregivers, while Eswatini while Eswatini included both caregivers and adults living with HIV; however, the smaller sample size in Eswatini limited the depth of subgroup and cross-country comparisons. Third, the study team in all three countries was also unable to return to participants to validate findings and ensure accuracy. Another limitation is that Microsoft Excel was used as the primary tool for data tabulation, without the support of specialized qualitative data analysis software. Taken together, these limitations may affect the generalizability and transferability of the results, as well as the depth and balance of the conclusions across different participant groups and contexts. The results should therefore be interpreted with caution, particularly when making comparisons between countries or demographic groups. Future studies would benefit from a more balanced sample, consistent inclusion criteria across all sites, the use of qualitative analysis software, and the incorporation of participant validation to enhance credibility.

Conclusion

Our findings suggest that, despite most participants having limited experience with SbPs and knowledge of Sb-TBD, they demonstrated a high willingness to provide stool samples, driven by their desire to recover, trust in the healthcare system, and the non-invasive nature of the procedure. However, their limited experience with this method may affect their ability to properly collect stool samples and may contribute to misconceptions that stool samples are not used for TB diagnosis. Further, hesitancy or refusal to provide samples in some participants could be influenced by factors such as transportation costs, challenges in providing stool on demand, feelings of disgust when handling stool, and local beliefs surrounding stool. In some cases, due to the perceived unpleasantness of stool and direct association of sputum with TB, patients may prefer sputum-based procedures over Sb-TBD. This collective evidence highlights the importance of including clear instructions for stool sample collection within the collection kits, alongside community-based interventions to increase awareness and understanding of Sb-TBD.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CISM

Centro de Investigação em Saúde da Manhiça

COREQ

Consolidated Criteria for Reporting Qualitative Research

EIs

Exit- Interviews

HIV

Human immunodeficiency virus

MDR-TB

Multidrug-resistant TB

PLWH

People Living with HIV

IDIs

In-depth interviews

SbPs

Stool‑based Procedures

SBS

Social‑Behavioural Study

SSI

Semi-structured Interviews

Sb-TBD

Stool‑based TB Diagnostics

TB

Tuberculosis

WHO

World Health Organization

Author contributions

Conceptualization: AVL, SA, SH, AK WS, AM, CL, KM and AGLB. Data curation: AVL, JNK, BN, FD, FS, and KM. Formal analysis: AVL, JNK, BN, FS, HC, KM, and ALGB Funding acquisition: SA, SH, AK, WS, AM, CL, KM, and ALGB. Investigation: AVL, JN, BN, FD, FS, and KM. Methodology: AVL and KM. Project administration: AVL, JNK, BN, FD, FS, WS, KM, and ALGB. Resources: WS, AK, AM, SH, KM, and ALGB. Supervision: KM and ALGB Validation: AVL, HC, SA, BN, JN, FS, LCC, JE, SH, AK, WS, AM, CL, KM, and ALGB. Visualization: AVL, JNK, BN, FD, FS, HC, and KM. Writing – original draft: AVL. Writing – review & editing: AVL, SA, BN, JN, LCC, JE, SH, AK, WS, AM, CL, HC, FS, KM, and ALGB.

Funding

This study is part of the EDCTP2 Program (grant number RIA2018D-2511-Stool4TB) supported by the European Union, and supported by Global Health EDCTP3 (grant number 101145812) and its members.

Data availability

All data relevant to the present study are in the article. The data used for this article are of a qualitative nature in which participants shared personal data/information, which is difficult to anonymize completely. Further, participants have not provided informed consent for their data to be stored in a public repository. Given these reasons and to guarantee confidentiality, we would rather not share our data in a public repository. Data are available from the authors upon reasonable request and with approval from the Executive and Scientific Committee of the Manhiça Health Research Centre. Request can be submitted via email: fundacao.manhica@manhica.net.

Declarations

Ethics approval and consent to participate

This study was conducted according to the protocol, the Declaration of Helsinki, as well as other locally relevant regulations. Ethical approval for the study was obtained from the Manhiça Health Research Centre’s Institutional Review Board in Mozambique (Ref. CISM-014–2021), the Makerere University Biomedical Research and Ethics Committee in Uganda (Ref. SBS-2022-151), and the Uganda National Council for Science and Technology (UNCST Ref. SS1492ES). In Eswatini, approval was granted by the Eswatini Health and Human Research Review Board (Ref. EHHRRB026/2025). Written informed consent was obtained from all participants. In case of illiteracy, participants were asked to provide their thumbprint in the companionship of an impartial witness who signed the consent form guaranteeing that respondents had been informed and voluntarily accepted to participate in the study. Participants were informed that they could withdraw from the study or stop the interview at any time and without any implication. The interviews were audio recorded with the permission, and according to the comfort of the respondents.

Consent for publication

No applicable.

Competing interests

The authors declare no competing interests.