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. 2022 Oct 4;17(10):e0264103. doi: 10.1371/journal.pone.0264103

The Simple One-step stool processing method for detection of Pulmonary tuberculosis: A study protocol to assess the robustness, stool storage conditions and sampling strategy for global implementation and scale-up

Petra de Haas 1,*, Bazezew Yenew 2, Getu Diriba 2, Misikir Amare 2, Andrii Slyzkyi 1, Yohannes Demissie 3, Bihil Sherefdin 3, Ahmed Bedru 3, Endale Mengesha 3, Zewdu Gashu Dememew 3, Abebaw Kebede 2,4,5, Muluwork Getahun 2, Edine Tiemersma 1, Degu Jerene 1
Editor: Padmapriya P Banada6
PMCID: PMC9531811  PMID: 36194578

Abstract

Background

The Xpert MTB/RIF Ultra (Xpert-Ultra) assay provides timely results with good sensitivity and acceptable specificity with stool specimens in children for bacteriological confirmation of tuberculosis (TB). This study aims to optimize the Simple One-Step (SOS) stool processing method for testing stool specimens using the Xpert-Ultra in children and adults in selected health facilities in Addis Ababa, Ethiopia. The study is designed to assess the robustness of the SOS stool method, to help fine-tune the practical aspects of performing the test and to provide insights in stool storage conditions and sampling strategies before the method can be implemented and scaled in routine settings in Ethiopia as well as globally.

Methods and design

The project “painless optimized diagnosis of TB in Ethiopian children” (PODTEC) will be a cross sectional study where three key experiments will be carried out focusing on 1) sampling strategy to investigate if the Xpert-Ultra M. tuberculosis (MTB) -positivity rate depends on stool consistency, and if sensitivity can be increased by taking more than one stool specimen from the same participant, or doing multiple tests from the same stool specimen, 2) storage conditions to determine how long and at what temperature stool can be stored without losing sensitivity, and 3) optimization of sensitivity and robustness of the SOS stool processing method by varying stool processing steps, stool volume, and storage time and conditions of the stool-sample reagent mixture. Stool specimens will be collected from participants (children and adults) who are either sputum or naso-gastric aspiration (NGA) and/or stool Xpert-Ultra MTB positive depending on the experiment. Stool specimens from these participants, recruited from 22 sites for an ongoing related study, will be utilized for the PODTEC experiments. The sample size is estimated to be 50 participants. We will use EpiData for data entry and Stata for data analysis purposes. The main analyses will include computing the loss or gain in the Xpert-Ultra MTB positivity rate and rates of non-determinate Xpert-Ultra test results per experiment compared to the Xpert-Ultra MTB result of stool processed according to the published standard operating procedures for SOS stool processing. The differences in the MTB positivity rate by regarding testing more than one sample per child, and using different storage, and processing conditions, will be also compared to the baseline (on-site) Xpert-Ultra result.

Introduction

Approximately 1.09 million children globally fall ill with tuberculosis (TB) each year, of whom only 399.000 are notified [1]. Every day, nearly 700 children die from TB, 80% of them before reaching their fifth birthday. Treatment exists that could prevent nearly all these deaths, but less than 5% of children get treatment as childhood TB is difficult to diagnose [2].

Recent WHO guidance on diagnosis and management of TB in children and adolescents recommends stool as non-invasive primary diagnostic specimen for testing with Xpert and Xpert Ultra for a diagnosis of TB among children [3, 4].

This new recommendation was guided by a recent systematic review, which showed that for stool, Xpert Ultra sensitivity against microbiological reference standard (MRS) was 53% in children aged 0 to 9 years, the sensitivity being higher in children younger than 1 year (65%), and lower in those aged 1 to 4 years (43%). Specificity was 96% to 98% [5]. Together with the Global laboratory initiative (GLI), WHO recently provided recommendations for the stool processing methods to be used, as there is a large variety on protocols for stool processing, with differences in reagents and methods of homogenization, filtering, and other steps leading to more complexity and a high heterogeneity in sensitivity [6]. One of the two stool processing methods recommended by WHO/GLI is the Simple One-Step (SOS) stool processing method, which is developed by KNCV Tuberculosis Foundation (KNCV) in collaboration with Ethiopian Public Health Institute (EPHI) [6, 7]. The SOS stool method uses similar steps as sputum Xpert and Xpert-Ultra testing and does not require additional materials or equipment other than an applicator to pick the correct stool amount for testing [6, 7]. Since the method is as simple as sputum testing, it can be performed at any site where a GeneXpert instrument is functional after providing minimal training to the staff involved in Xpert or Xpert-Ultra testing [6, 7].

To gain more knowledge and in-depth experience on how the SOS stool processing method with Xpert-Ultra would behave if included in the routine diagnostic pathway for (childhood) TB and rolled out under the national TB program, we aim to further test and optimize the SOS processing method for the detection of TB in stool by Xpert-Ultra and its ability to tolerate perturbations (robustness). The study will also help to fine-tune standard operating procedures (SOPs) for the SOS stool method.

Materials and methods

Study setup and period

This will be a cross-sectional study that will consist of a series of experiments on consecutive stool specimens collected from children and adults that are either sputum/NGA and/or stool Xpert-Ultra MTB positive. The study will be conducted in multiple health care facilities (>20) in Addis Ababa, Ethiopia. The facilities have a relatively high number of TB patients and have experience with participation in research. Children are being recruited for another related study, that assesses the diagnostic accuracy of Xpert stool testing using the SOS stool processing method, called Alternatives to Sputum Testing for Tuberculosis in Indonesia and Ethiopia (ASTTIE), see Fig 1. Therefore, MTB positive children who will be identified in the ASTTIE study will also be used in the current study (PODTEC study). We will also recruit adults with MTB detected in sputum from the same facilities. The study was originally planned to be carried out till the end of 2020. However, due to the COVID-19 pandemic, the study period has been extended.

Fig 1. Study flow diagram representing steps from inclusion of adults and children to arrival of the stool samples at EPHI and their allocation to the different experiments.

Fig 1

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* Site of action is depicted with blue background boxes; and stool samples are in yellow boxes. Abbreviations used: ASTTIE; Alternatives to Sputum Testing for Tuberculosis in Indonesia and Ethiopia, PODTEC: Painless Optimized Diagnosis of TB in Ethiopian Children, NTRL: National TB Reference Laboratory, NGA: Naso-Gastric Aspiration, MTB: M.tuberculosis, GX; GeneXpert, Exp.: Experiment.

Study population

Children aged ≤10 years from the ASTTIE study who are sputum/NGA and/or stool Xpert-Ultra MTB positive, and consecutive sputum Xpert MTB-positive adults presenting in the selected health facilities will constitute the study population. Eligible persons, or their caregivers will be requested to sign (parental) consent or assent, depending on the age of the participant. The exclusion criteria include being critically sick i.e., those who are in coma, terminally ill due to chronic debilitating co-morbidities, or other conditions determined to be “critical” by the treating physician, being on TB treatment for longer than 5 days at the time of recruitment, and refusal to sign the informed consent.

Participant enrolment and stool collection

For children, the facility coordinator from the ASTTIE project will daily retrieve stool Xpert-Ultra results from the study sites and checks for eligibility. Parents of eligible children and eligible adults will be asked for informed consent for participation in this study, see S1 Appendix. For children, this is an additional consent to the consent already provided for the ASTTIE study. After enrolment in the study, for children, the remainder of the initial stool specimen (stool 1) will be collected and transported to EPHI. Participants will be provided with two (children) or three (adults) large stool containers to allow collection of at least 30 grams of stool on consecutive days. They will be instructed on how to collect and store the specimens till delivery at the site. Three appointments will be made to submit the additional stool specimens. When the specimens are submitted, information will be collected on the stool submission form S2 Appendix about the date and time of collection at the household, storage conditions at the household and during transport and date and time of arrival at the site. To maximize the likelihood of finding MTB in the stool, the additional stool specimens should be collected within 5 days after the participant’s TB treatment starts. Participants will be reimbursed for travel costs.

The stool specimens will be kept in a cold chain at the site until they are transported to EPHI on the same day. The site will inform the study coordinator which will assign a dedicated transporter for this research purpose. The time between contacting the study coordinator and the pick-up of the specimens is expected to be within 2 hours after delivery on-site, which means that the specimens should arrive on the same day of collection at EPHI. After arrival at EPHI, the dedicated laboratory technician will be ready to receive and register the specimens and start the cascade of experiments as shown in the specimen flow diagram Fig 1.

Design of the experiments

In total three experiments are designed and the lay out is depicted in Figs 2 and 3.

Fig 2. Overview of experiments.

Fig 2

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Arrows indicate what experiments are done with the different stool specimens. Note that for adult participants, in principle all experiments will be done on all three stool samples, provided that enough stool is collected per bowel movement. Abbreviations used: MTB, M. tuberculosis; N, North; S, South; E/W, East or West; Exp, experiment; RT, room temperature; H, hours; SR, sample reagent; g, gram.

Fig 3. Overview of assigning stool to the experiments outlined in Fig 2.

Fig 3

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Note that for adults, in principle, all experiments will be conducted if there is enough stool available per sample. Abbreviations used: ASTTIE; Alternatives to Sputum Testing for Tuberculosis in Indonesia and Ethiopia, MTB: M. tuberculosis, GX; GeneXpert, Exp.: Experiment; g, gram: ml, milliliter. Explanation of experiment numbers: Experiment 1: Stool sampling strategy; Experiment 2: Stool sample storage conditions; Experiment 3a: incubation time and shaking method; Experiment 3b: time and temperature for keeping the processed stool-sample reagent mixture; Experiment 3c: stool volume.

Experiment 1: Stool sampling strategy

This experiment will investigate if, and by how much, the positivity rate of Xpert-Ultra on stool increases when more than one specimen from the same participant is tested. It also indicates how homogeneous the Mycobacteria are distributed within the stool specimens and across different stools. Furthermore, it will provide insight in repeat testing if required due to unsuccessful test result, whether to advise to perform the repeat test from the same stool or from a fresh stool specimen. This will be done by testing three stools from the same participant collected during consecutive days (Experiment 1a) as well as three aliquots (North, South and East/West) from the same stool specimen (Experiment 1b). For children, two aliquots will be collected from the first stool specimen Fig 1. Aliquots will be tested using Xpert-Ultra, totaling a maximum of nine Xpert-Ultra tests per participant.

Experiment 2: Stool sample storage conditions

This experiment will investigate how long and under which conditions stool can be kept without losing sensitivity to detect TB on Xpert-Ultra or increasing rates of unsuccessful tests. This is done by testing multiple aliquots taken from a known Xpert-Ultra MTB-positive stool specimen after storing aliquots from that Xpert-Ultra MTB-positive stool at three temperatures; a) refrigerator 2–8°C, b) room temperature 20–22°C, and c) incubator 37°C and at four time periods; 2, 3, 5 and 10 days. These experiments will be done using aliquots remaining from the second and third stool specimens collected for Experiment 1. These stool specimens are expected to be larger in size and probably have the shortest time interval between collection at the site and preparation at EPHI.

Experiment 3: Optimization and evaluating robustness of the SOS stool processing method

This experiment consists of a series of sub-experiments that will investigate if the SOS stool processing method can be further optimized to increase its recovery rate to detect TB. Although the SOS stool processing method is simple and contains minimal processing steps, certain steps might still be adapted to see if this influences the test’s sensitivity. This is done by testing multiple aliquots taken from a known Xpert-Ultra MTB-positive stool specimen processed using slightly different approaches. The first sub-experiment (3a) varies the incubation time and shaking method during the processing of stool. The second sub-experiment (3b) assesses the optimum and maximum time and temperature for keeping the processed stool-sample reagent mixture before Xpert-Ultra testing is conducted on the different incubation steps. The third sub-experiment (3c) assesses the optimum and maximum stool volume.

Stool consistency and bacterial load are two important factors that might influence the outcome of the experiments. Therefore, specimens with different consistency and bacterial load will be included in all experiments.

If the Xpert-Ultra result is unsuccessful (i.e., the result is “invalid” or “error”), the test will NOT be repeated as this is part of the study outcome.

The SOS stool method’s comprehensive instructions can be found on KNCV website [8]. In S3 Appendix a schematic overview of the SOS stool method is provided. Depending on the stool consistency the protocol for solid stool or liquid stool is followed.

Variables and outcome measures

The primary outcome measures will be the rate of Xpert-Ultra indeterminate test results. The secondary outcome measures will be the Xpert-Ultra MTB (semi-)quantitative result and positivity rate of stool specimens processed using the SOS stool processing method. Values of these outcomes obtained under the different experimental conditions will be compared to the baseline (on-site) Xpert-Ultra MTB positive test result of stool processed using the SOS stool processing method and to the values obtained under per current protocol experimental conditions [6, 7].

Sample size

At the selected health facilities, a maximum of 750 children with presumptive TB will be enrolled in ASTTIE during the recruitment period of this study. With Xpert-Ultra MTB-positivity rate of 5%, up to 32 children will be available for the optimization exercises. We aim to supplement this with up to 50 participants by also recruiting adults from the health facilities participating in ASTTIE, as described above, as the SOS stool test with Xpert has shown to be also useful for adults who cannot spontaneously expectorate sputum [9]. Thus, for the experiments, we will have stool specimens for around 50 individuals available, totaling a maximum of 150 stool specimens.

Fig 4 indicates the minimum rates of conversion (Xpert MTB- to Xpert MTB+), respectively reversion (Xpert MTB+ to Xpert MTB-), that can be detected with statistical significance with this sample size.

Fig 4. Required sample size as estimated using McNemar’s two-sample paired proportions test using Stata SE v.

Fig 4

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17.0, assuming discordance in pairs of stool aliquots.

Analysis plan and data collection

Data collected will include age and sex of the participant, TB suggestive symptoms and TB contact history, Xpert-Ultra result for the initial stool (children only) and sputum/NGA specimen, date, and place (participant’s home or health facility) of stool collection, date of stool receipt at the NTRL, stool storage and transport temperature until receipt at the NTRL, and stool consistency. Detailed information on the experiments’ conditions will also be collected as well as the cycle threshold (Ct) values for all probes and error codes in case of errors.

Data entry, storage, and management

Each stool specimen will be submitted to EPHI together with a stool submission form S2 Appendix. Details when conducting the experiment are collected on the experiment form S4 Appendix. The forms are stored at EPHI. All data will be entered into pre-structured EpiData files (EpiData version 3.1; www.epidata.dk). A random 10% of the data will be re-entered in a separate file to check the quality of data entry. If more than 3% of errors are found in key variables (experiment conditions and Xpert result), full double data entry will be conducted.

Data interpretation

The main study outcome is the semi-quantitative Xpert Ultra result as provided by the GeneXpert instrument (trace, very low, low, medium, high, error, invalid or no result), interpreted as per the manufacturer’s instructions. The individual probes’ Ct values are the main quantitative study outcomes. We consider higher Ct values to represent lower bacillary loads, as these indicate that more PCR cycles are needed to reach the threshold of MTB detected. Error codes will be interpreted following the manufacturer’s guidance to understand the likely cause of the error.

Statistical analysis

Statistical analysis will be performed by the STATA/SE (version 15; StataCorp) statistical software package. The Xpert-Ultra stool result from each aliquot will be compared with the baseline Xpert-Ultra. Trends in the proportion of specimens being MTB-positive and the proportion of specimens with unsuccessful results over e.g., increasing storage time or temperature, or increasing amounts of stool added, will be analyzed using Wilcoxon-like test for trend of across ordered groups using nptrend [10]. Logistic regression will be applied to assess factors associated with stool MTB positivity and unsuccessful test outcomes. We will assess if there is indication that stool specimens are nested into individual participants, just as aliquots of one stool specimen may be nested into that stool specimen, by comparing the outcomes of simple (multivariate) logistic regression to the outcomes of multilevel mixed-effects logistic regression using the likelihood-ratio test to determine the best model fit.

Ethical considerations

The study obtained ethical clearance from the Review Boards of the Ethiopian Public Health Institute (EPHI-IRB) (Protocol no EPHI-IRB-234-2020). The project will follow the routine procedure of patients’ recruitment into studies, follow-up, and analysis as well as drawing of conclusions. Informed parental consent will be obtained from the children’s legal guardians. Participants’ information will be kept confidential, and the digital files used for analysis will only have the PODTEC laboratory code and ASTTIE unique person identification code (UPIC) and will not contain any names or other personal identifying information of the participant. Participants’ information will be kept confidential, and the digital files used for analysis will only have the PODTEC laboratory code and the ASTTIE UPIC and will not contain any names or other personal identifying information of the participant. The study results will be shared with the national TB program and stakeholders to the benefit of further roll out of the test in a routine Ethiopian setting. The results will also be disseminated in peer-reviewed scientific journals.

Discussion

This is the first study protocol to be published in which the sampling strategy and robustness of a stool processing method will be investigated. Based on the experiment’s findings, certain steps in the current SOP of the SOS stool method might be adjusted. The experiments will be performed using specimens from bacteriologically confirmed TB patients, so for the patients for whom the test will be used in practice in a country with a relatively high TB burden. Stool specimens will not be spiked with MTB-complex bacteria as in some other studies [11, 12]. We expect that the MTB distribution is different in the stool specimens from TB patients than in the spiked stools. Moreover, we will include children who have mostly have paucibacillary TB and who will benefit most from stool Xpert or Xpert-Ultra testing, as they cannot easily provide sputum. The study population will be drawn from locations where the test is expected to be conducted in the future, providing more realistic insights in the possibility of implementing the method in routine and collection of multiple specimens.

The experiments are based on the controlled simulation of plausible scenarios, i.e. situations that can occur in practice, such as long transit times at high temperatures, long contact time of stool with the sample reagent before Xpert-Ultra testing, variation in the stool portion size picked for processing, and variation in stool processing. The outcome will provide insights of the robustness of the method and will show how far certain steps can be stretched. It will provide practical outcomes that will enable the laboratory personnel and healthcare professionals involved in the stool testing to implement the most optimal protocol.

The main results will be presented both at local and international scientific meetings. The results will also be disseminated in the form of peer reviewed publications and as policy briefs. Key audiences for the dissemination will include global scientific advisory group members, local technical advisory committee (TAC) members and NTP. Study host communities will also be informed about the key results of the study through appropriate popular media.

Supporting information

S1 Appendix

(PDF)

Click here for additional data file. (123.1KB, pdf)
S2 Appendix

(PDF)

Click here for additional data file. (170.1KB, pdf)
S3 Appendix

(TIF)

Click here for additional data file. (257.6KB, tif)
S4 Appendix

(PDF)

Click here for additional data file. (191.8KB, pdf)

Acknowledgments

We would like to acknowledge the Ethiopian Public Health Institute and KNCV Tuberculosis Foundation. We would also like to thank all healthcare facilities that will participate in the study by recruiting the study participants. We would further like to acknowledge Mamush Sahile from KNCV Ethiopia for his assistance in the study.

Data Availability

No datasets were generated or analysed during the current study. All relevant data from this study will be made available upon study completion.

Funding Statement

This study protocol is part of the PODTEC (painless optimised diagnosis of TB in ethiopian childern) study and is funded by a private funder. The funder had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

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Decision Letter 0

Padmapriya P Banada

23 May 2022

PONE-D-22-03274The Simple One-step stool processing method for detection of Pulmonary tuberculosis: a study protocol to assess the robustness, stool storage conditions and sampling strategy for global implementation and scale-upPLOS ONE

Dear Dr. de haas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The study protocol is presented well and well throughout. But it does need improvement and clarity. As suggested by the reviewer the flow charts could be simple. I hope the comments would help you improve the manuscript and I look forward for your resubmitted manuscript.

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Additional Editor Comments:

Dear authors,

The study protocol is presented well and well thoughtout. But it does need improvement and clarity. As suggested by the reviewer the flow charts could be simple. I hope the comments would help you improve the manuscript and I look forward for your resubmitted manuscript.

Best

Priya

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #1: The protocol proposed by De Haas and colleagues aims to assess different components of stool collection and processing which could influence detection of MTB by Xpert Ultra. The flow charts are rather complex but have clearly been thought through carefully.

My main points of criticism are the following:

- The background information needs to be more balanced to accurately summarize current evidence of different stool processing methods for TB diagnosis (see details below)

- I am uncertain about the sample size calculation and whether the estimated sample size will be able to detect a meaningful difference between the experiments and the baseline method.

Abstract

General: The abstract uses the terms “samples” and “sampling” loosely to mean specimen, statistical sampling and testing different parts of a sample. I recommend reviewing the entire abstract to use more accurate terminology.

Other:

Line 21: “Xpert MTB/RIF Ultra (Xpert-Ultra) provides timely results with good sensitivity and acceptable specificity with stool samples in children for bacteriological confirmation of tuberculosis (TB).” There are actually no data on Ultra on stool in children- only Xpert MTB/RIF.

Line 41: Reword: “The sample size is estimated to be 50 participants.”

Line 43-47: Reword: “We will use EpiData for data entry and Stata for data analysis purposes. The main analyses will include computing the loss or gain in the Xpert-Ultra MTB positivity rate compared to ..., and rates of unsuccessful test results. The differences in the positivity rate by regarding testing more than one sample per child, and using different storage, and processing conditions, will be compared to the baseline (on-site) Xpert-Ultra result.”

Introduction

WHO has made no recommendation on stool as a sample for use with Ultra due to lack of evidence. The recommendation for stool is for Xpert MTB RIF only, and it is based on low certainty of evidence.

Lines 71-72: “This shows that there is a lack of standardized stool preparation and testing protocols and warrants the optimization and standardization of the stool processing methods that can be used at the decentralized level.”

Suggest rewording: “The lack of standardized stool preparation and testing protocols warrants the optimization and standardization of stool processing methods that can be used at the decentralized level

Lines 81-82: I suggest rewording: A pilot study conducted in multiple laboratories across Ethiopia demonstrated acceptably low rates of unsuccessful test results (6%).

Lines 83-84: “Furthermore, a head-to-head comparison study, in which the SOS method is compared to other stool processing methods showed similar sensitivity and specificity.” I think it is rash to make this statement based on a laboratory spiking study which tested a small number of samples. Even so, the Walters-centrifugation method appeared superior in detecting BCG at lower concentrations, which is relevant in the case of young children/infants, who have more extreme forms of paucibacillary disease and for whom stool is more attractive than for older children and adolescents. I would favour a more balanced summary of the quoted study.

Methods

Enrolment: I would strongly advise enriching for young children and infants <2 years of age, as this is the group for whom stool-based diagnosis is most relevant. This is also the group more likely to have very low bacillary concentrations in sputum and hence stool. A stool-based method that can detect TB in older children with adult-type cavitary TB and other forms of TB with higher bacillary loads (many of these children will be able to produce sputum) is not as relevant.

Lines 192-196 : The sentence is grammatically incorrect. Please clarify if the primary outcome is the Ultra positivity rate of the index experiment vs the on-site Ultra stool result? Is the secondary outcomes measure also a comparison of the index experiment vs on-site Ultra stool? Please edit accordingly.

I am not an expert in statistics, but I have some concerns regarding the sample size calculation and the effect size that such a sample size will be able to achieve. In the footnote to the figure, the authors say that the SS calculations do not take into account for correlation between stool samples from the same individual. Surely, this should be considered? Secondly, even assuming that all the samples are collected, the minimum difference in detection (from negative to positive) that will be measured with statistical significance is 10%. Does that mean that ANY of the experiments will be able to detect 10% more TB than the baseline test? Is that meaningful? Should the sample size not be calculated to achieve an meaningful increase in detection for every experiment? As I am not an expert in this, I think the statistical methods should be reviewed by a statistician, and clarified for a non-expert readership.

Discussion

Lines 262-263. I know that other stool processing methods have undergone similar pre-clinical testing, but the protocols were not published. So rather say that this is the first protocol to be published...

Line 265: The experiments will actually be conducted on confirmed TB cases based on the inclusion criteria, not presumptive TB cases.

Line 266-267: There are a number of published studies assessing stool-based TB diagnosis which have used clinical samples (not only spiked samples).

Minor:

Line 70: rather quite- redundancy

Line 212: Suggest rewording: “Data collected will include age...”

Line 214: “of stool of collection”- delete second “of”

Reviewer #2: This study protocol is very elaborative and well designed. The authors here would like to emphasize on the need of a simple and standard protocol for Mtb diagnosis from stool samples. Their study protocol is easy to understand with enough supporting information. I would like to provide my concerns/suggestions listed below.

1. Which sample will be used as baseline? the stool or the sputum or both?

2. Will the samples be collected on two/three consecutive days from the study population or there will be some duration between two samples collected from the same person? My suggestion will be to include this information in the study protocol as well as in the consent form.

3. Are you going to enroll Mtb negative population for this study as a control group?

4. For experiment 2, to study the effect of storage conditions, you have not proposed to freeze any samples. I believe the addition of freezing as a stool storage temperature and then study the effect of freezing on Mtb detection using your proposed SOS method would add value to your studies and also help others in future.

**********

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PLoS One. 2022 Oct 4;17(10):e0264103. doi: 10.1371/journal.pone.0264103.r002

Author response to Decision Letter 0

4 Jul 2022

Feedback to the reviewers

Reviewer #1: The protocol proposed by De Haas and colleagues aims to assess different components of stool collection and processing which could influence detection of MTB by Xpert-Ultra. The flow charts are rather complex but have clearly been thought through carefully.

My main points of criticism are the following:

- The background information needs to be more balanced to accurately summarize current evidence of different stool processing methods for TB diagnosis (see details below)

- I am uncertain about the sample size calculation and whether the estimated sample size will be able to detect a meaningful difference between the experiments and the baseline method.

� We thank the reviewer for these considerations and refer to our answers below, in which we have attempted to address these concerns as much as follows.

Abstract

General: The abstract uses the terms “samples” and “sampling” loosely to mean specimen, statistical sampling and testing different parts of a sample. I recommend reviewing the entire abstract to use more accurate terminology.

� We agree with the reviewer that the use of the word “sample” for biological specimens and for statistical sample size might lead to confusion among readers. Therefore, we consistently changed the word “sample” to “specimen” when it was referring to a biological specimen (either stool or NGA/sputum) throughout the manuscript. We believe that we already were consistent in using the word “aliquot” for testing different parts of the stool specimen. We left the word “sample” in for the following: 1) when referring to the sample reagent as Cepheid has given this name to their buffer; and 2) when referring to sample size, as these two words were always used in combination. We kept in the term “sampling strategy” as these two words were always used in combination.

Other:

Line 21: “Xpert MTB/RIF Ultra (Xpert-Ultra) provides timely results with good sensitivity and acceptable specificity with stool samples in children for bacteriological confirmation of tuberculosis (TB).” There are actually no data on Ultra on stool in children- only Xpert MTB/RIF.

� The reviewer is correct that at the time the protocol was drafted there was no published data on the use of Xpert-Ultra on stool. However, there was un-published data availble from ongoing studies conducted by KNCV and by other reasearchers that provided promising results. This made us decide to use Xpert-Ultra within this study, as this will also be expected to be used in future routine practise. Meanwhile, data has been published and recommendations have been provided by WHO for the use of Xpert-Ultra. We changed the introduction accordingly. Note that we added the recently published updated guideline on the management of tuberculosis in children and adolescents as a reference and the GLI practical manual on stool processing methods in which the SOS stool method is one of the two recommended methods.

Line 41: Reword: “The sample size is estimated to be 50 participants.”

� Indeed, this sentence was phrased incorrectly. We changed the sentence as suggested to “The sample size is estimated to be 50 participants.”

Line 43-47: Reword: “We will use EpiData for data entry and Stata for data analysis purposes. The main analyses will include computing the loss or gain in the Xpert-Ultra MTB positivity rate compared to ..., and rates of unsuccessful test results. The differences in the positivity rate by regarding testing more than one sample per child, and using different storage, and processing conditions, will be compared to the baseline (on-site) Xpert-Ultra result.”

� We thank the reviewer for this suggestion, it clarifies better what our standard is. We changed the text as suggested: The main analyses will include computing the loss or gain in the Xpert-Ultra MTB positivity rate and rates of non-determinate Xpert-Ultra test results per experiment compared to the Xpert-Ultra MTB result of stool processed according to the published standard operating procedures for SOS stool processing. The differences in the MTB positivity rate by regarding testing more than one sample per child, and using different storage, and processing conditions, will be also compared to the baseline (on-site) Xpert-Ultra result”.

Introduction

WHO has made no recommendation on stool as a sample for use with Ultra due to lack of evidence. The recommendation for stool is for Xpert MTB RIF only, and it is based on low certainty of evidence.

� See our reply to the comment above in which we explained that evidence on the use of Xpert-Ultra on stool recently became available through the published updated guideline on management of tuberculosis in children and adolescents, we revised the text accordingly.

Lines 71-72: “This shows that there is a lack of standardized stool preparation and testing protocols and warrants the optimization and standardization of the stool processing methods that can be used at the decentralized level.”

Suggest rewording: “The lack of standardized stool preparation and testing protocols warrants the optimization and standardization of stool processing methods that can be used at the decentralized level

� We agree with suggestion of the reviewer and changed the text to: “The lack of standardized stool preparation and testing protocols warrants the optimization and standardization of stool processing methods that can be used at the decentralized level” as suggested.

Lines 81-82: I suggest rewording: A pilot study conducted in multiple laboratories across Ethiopia demonstrated acceptably low rates of unsuccessful test results (6%).

� Thank you for this suggestion. As we decided to update the introduction section with new presented in the WHO guidelines (see above), we decided to remove this sentence from the introduction section.

Lines 83-84: “Furthermore, a head-to-head comparison study, in which the SOS method is compared to other stool processing methods showed similar sensitivity and specificity.” I think it is rash to make this statement based on a laboratory spiking study which tested a small number of samples. Even so, the Walters-centrifugation method appeared superior in detecting BCG at lower concentrations, which is relevant in the case of young children/infants, who have more extreme forms of paucibacillary disease and for whom stool is more attractive than for older children and adolescents. I would favour a more balanced summary of the quoted study.

� Thank you for pointing out that we referred to the wrong paper. Jasumback’s paper was indeed using spiked samples and did not describe a head-to-head comparison study. Instead, we had meant to refer to the head-to-head comparison study done by FIND and TB-Speed, which is included in the most recent Guideline. We have corrected the reference.

Methods

Enrolment: I would strongly advise enriching for young children and infants <2 years of age, as this is the group for whom stool-based diagnosis is most relevant. This is also the group more likely to have very low bacillary concentrations in sputum and hence stool. A stool-based method that can detect TB in older children with adult-type cavitary TB and other forms of TB with higher bacillary loads (many of these children will be able to produce sputum) is not as relevant.

� We agree with the reviewer that TB diagnosis based on stool instead of sputum is most relevant for young children as these cannot produce sputum. Our experience is that children up to 10 years old experience difficulty with sputum production. There are, however, also adult groups, such as PLHIV, that cannot easily produce a spontaneous sputum sample and for these groups, stool can be an alternative sample, as we recently showed in a pilot implementation project in Vietnam. We have now included this reference in lines 201.

Lines 192-196: The sentence is grammatically incorrect. Please clarify if the primary outcome is the Ultra positivity rate of the index experiment vs the on-site Ultra stool result? Is the secondary outcomes measure also a comparison of the index experiment vs on-site Ultra stool? Please edit accordingly.

� We thank the reviewer for remarking this. We have changed the text as follows: “The primary outcome measures will be the rate of Xpert-Ultra indeterminate test results”. The secondary outcome measure will be the Xpert-Ultra MTB (semi-)quantitative result and positivity rate of stool specimens processed using the SOS stool processing method. Values of these outcomes obtained under the different experimental conditions will be compared to the baseline (on-site) Xpert-Ultra MTB positive test result of stool processed using the SOS stool processing method and to the values obtained under per current protocol experimental conditions (6, 7).

I am not an expert in statistics, but I have some concerns regarding the sample size calculation and the effect size that such a sample size will be able to achieve. In the footnote to the figure, the authors say that the SS calculations do not take into account for correlation between stool samples from the same individual. Surely, this should be considered? Secondly, even assuming that all the samples are collected, the minimum difference in detection (from negative to positive) that will be measured with statistical significance is 10%. Does that mean that ANY of the experiments will be able to detect 10% more TB than the baseline test? Is that meaningful? Should the sample size not be calculated to achieve an meaningful increase in detection for every experiment? As I am not an expert in this, I think the statistical methods should be reviewed by a statistician, and clarified for a non-expert readership.

� Several of the authors are senior epidemiologists and thus have some statistical education, but we are consulting a statistician for this now. We have used the Statulator (http://statulator.com/SampleSize/ss2PP.html#) which calculates the sample size needed for paired proportions, and then calculated how many observations are needed if x% of the population shifts from MTB+ to MTB-, or, alternatively from a meaningful result (MTB+/MTB-) to an indeterminate result (error, invalid, no result). Thus, this considers the fact that one measurement from the same person (or stool) is paired to another measurement from that same person. Hence, the footnote under the figure is not correct and we removed the footnote.

We do however agree that the sample size may be too small for the sampling experiment as the positivity rate may not drop/increase with 20% from baseline experiment (or per-protocol experiment). It may probably be sufficient for the robustness experiments, as (initial) indeterminate rates tend to be quite high when deviating from the protocol.

Discussion

Lines 262-263. I know that other stool processing methods have undergone similar pre-clinical testing, but the protocols were not published. So rather say that this is the first protocol to be published...

� We are pleased the reviewer is pointing this out, we added “to be published” to the sentence as suggested.

Line 265: The experiments will actually be conducted on confirmed TB cases based on the inclusion criteria, not presumptive TB cases.

� This is indeed incorrect, and we thank the reviewer for this correction. We changed presumptive TB cases to “bacteriologically confirmed TB patients”

Line 266-267: There are a number of published studies assessing stool-based TB diagnosis which have used clinical samples (not only spiked samples).

� The reviewer is correct. Therefore, we have deleted the word “uniquely” and changed the sentence as follows: Stool samples will not be spiked with mycobacteria as in some other studies (9), (12).

Minor:

Line 70: rather quite- redundancy

� We have deleted the word “rather”.

Line 212: Suggest rewording: “Data collected will include age...”

� We have changed the sentence accordingly.

Line 214: “of stool of collection”- delete second “of”

� We have deleted the word “of” between “stool” and “collection”.

Reviewer #2: This study protocol is very elaborative and well designed. The authors here would like to emphasize on the need of a simple and standard protocol for Mtb diagnosis from stool samples. Their study protocol is easy to understand with enough supporting information. I would like to provide my concerns/suggestions listed below.

We thank the reviewer for this positive feedback and are pleased to hear that the protocol was easy to understand. We provided the answers to the concerns and suggestions below.

1. Which sample will be used as baseline? the stool or the sputum or both?

� For the baseline at enrolment, we used both the Xpert-Ultra result of the stool and the sputum specimen depending on the comparison that will be made.

2. Will the samples be collected on two/three consecutive days from the study population or there will be some duration between two samples collected from the same person? My suggestion will be to include this information in the study protocol as well as in the consent form.

� We are pleased that the reviewer is pointing this out as this is indeed an essential detail. We changed the sentence in the method section as follows: Participants will be provided with two (children) or three (adults) large stool containers to allow collection of at least 30 grams of stool on consecutive days. This will indeed be explained to the participants upon providing information about the study. Please note however, that in practice, we will depend on the participant’s willingness to travel back to the facility on consecutive days. For practical reasons, we therefore will allow any sample that is collected within 5 days after starting TB treatment.

3. Are you going to enroll Mtb negative population for this study as a control group?

� We like to thank the reviewer for this suggestion, as we have been doubting taking that approach. However, adding these samples would massively increase the sample size and the cost of the study as MTB won’t be found in the great majority of presumptive TB patients, especially when it concerns children. Besides, the MTB-negative population might only provide limited insights in the main objective of assessing the robustness of the SOS stool method and stool storage and transit conditions.

Although we agree that there would be benefit in including MTB-negative persons for the sampling strategy experiment, we assume that the chance of finding MTB in one of the aliquots of an (initially) MTB-negative person is very low (<<10%) and we therefore do not include subjects who are negative on both stool and sputum. However, as we will include patients with either sputum/GA or stool MTB positive, we might have some participants for whom the initial stool specimen is MTB negative.

4. For experiment 2, to study the effect of storage conditions, you have not proposed to freeze any samples. I believe the addition of freezing as a stool storage temperature and then study the effect of freezing on Mtb detection using your proposed SOS method would add value to your studies and also help others in future.

� We agree with the reviewer, and we had originally planned to include storage in freezer as one of the options. However, logistical constraints made us to remove that option. First, our partner in Ethiopia has limited space in their freezers, for which use we also would have to pay. Second, there is limited funding available for this study. Third, the amount of stool collected for the experiments will limit the number of aliquots that we can take from these stools for the experiments. In routine practice, stool storage before processing most likely will occur in the refrigerator or at ambient temperature (which is between 20-25°C in Ethiopia but may be well above 30°C in other countries), rather than in a freezer. That’s why we chose to store stool at (4-6°C, RT, and 36°C). However, to get some insight in the effect of storage of stool in a freezer, we have included in protocol that left-over stool (if any) should be stored in the -20°C freezer.

Attachment

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Decision Letter 1

Padmapriya P Banada

26 Jul 2022

PONE-D-22-03274R1The Simple One-step stool processing method for detection of Pulmonary tuberculosis: a study protocol to assess the robustness, stool storage conditions and sampling strategy for global implementation and scale-upPLOS ONE

Dear Dr. de haas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

 The above manuscript is greatly improved int the revised version. I am mostly satisfied with your response to the reviewers concerns. The protocol carries much better clarity. However, right statistics do play a major role in determining the sample size and the analysis of the proposed study. I am not sure if the use of Statulator.com is enough for such studies. I do like to see the study established in consultation with an expert statistician, especially if enrolling a small number of MTB confirmed negative patients might help them establish their specificity. I understand the financial and resource limitations, but strongly recommend it, as this would be very important in understanding the application of the proposed method for routine use. Therefore I am recommending minor revision to the protocol.**********

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The above manuscript is greatly improved int the revised version. I am mostly satisfied with your response to the reviewers concerns. The protocol carries much better clarity. However, right statistics do play a major role in determining the sample size and the analysis of the proposed study. I am not sure if the use of Statulator.com is enough for such studies. I do like to see the study established in consultation with an expert statistician, especially if enrolling a small number of MTB confirmed negative patients might help them establish their specificity. I understand the financial and resource limitations, but strongly recommend it, as this would be very important in understanding the application of the proposed method for routine use. Therefore I am recommending minor revision to the protocol.

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Reviewers' comments:

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PLoS One. 2022 Oct 4;17(10):e0264103. doi: 10.1371/journal.pone.0264103.r004

Author response to Decision Letter 1

26 Aug 2022

We would like to thank the reviewers for their positive response on the revised version of the manuscript. It was nice to hear that the protocol has greatly improved. We herewith like to provide feedback to the last comment provided by reviewer 1

“The above manuscript is greatly improved int the revised version. I am mostly satisfied with your response to the reviewer’s concerns. The protocol carries much better clarity. However, right statistics do play a major role in determining the sample size and the analysis of the proposed study. I am not sure if the use of Statulator.com is enough for such studies. I do like to see the study established in consultation with an expert statistician, especially if enrolling a small number of MTB confirmed negative patients might help them establish their specificity. I understand the financial and resource limitations, but strongly recommend it, as this would be very important in understanding the application of the proposed method for routine use. Therefore, I am recommending minor revision to the protocol.”

Reply: Though not explicitly stated on their website, Statulator seems to make use of Mcnemar’s two-sample paired proportions test to estimate sample sizes. We consulted Dr L. Stuck at the university of Amsterdam, a statistician, epidemiologist and data scientist (https://www.aighd.org/people/logan-stuck/), who confirmed that this is the correct test to use for sample size calculations. For reproducibility, we repeated the sample size calculations in Stata using McNemar’s test. This can be done using the “power paired proportions” command. We have adapted the title of Figure 4 accordingly.

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Decision Letter 2

Padmapriya P Banada

12 Sep 2022

The Simple One-step stool processing method for detection of Pulmonary tuberculosis: a study protocol to assess the robustness, stool storage conditions and sampling strategy for global implementation and scale-up

PONE-D-22-03274R2

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Academic Editor

PLOS ONE

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Reviewers' comments:

Acceptance letter

Padmapriya P Banada

26 Sep 2022

PONE-D-22-03274R2

The Simple One-step stool processing method for detection of Pulmonary tuberculosis: a study protocol to assess the robustness, stool storage conditions and sampling strategy for global implementation and scale-up

Dear Dr. de haas:

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