Stool-based Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children at a teaching and referral hospital in Southwest Ethiopia.

BACKGROUND: Diagnosis of tuberculosis (TB) in children is challenging mainly due to the difficulty of obtaining respiratory specimen and lack of sensitive diagnostic tests. The objective of this study was to evaluate the diagnostic performance of Xpert MTB/RIF (Xpert here after) for the diagnosis of pulmonary TB (PTB) from stool specimen in children.
METHODS: A cross-sectional study was conducted among consecutively recruited children (less than 15 years old) with presumptive PTB at Jimma Medical Center, Ethiopia. One pulmonary specimen (expectorated sputum or gastric aspirate) was collected from each participant and tested for TB by Xpert and Lowenstein-Jensen (LJ) culture. In addition, one stool specimen per child was collected and tested by Xpert after a single step, centrifuge-free stool processing method adapted from KNCV TB Foundation. Diagnostic performance of Xpert was calculated with reference to LJ culture and to a composite reference standards (CRS) comprising of confirmed TB (positive by Xpert and/or culture) and unconfirmed TB (clinical diagnosis with improvement after anti-TB treatment).
RESULTS: A total of 178 children were enrolled; 152 of whom had complete microbiological results. Overall, TB was diagnosed in 13.2% (20/152) of the children with presumptive TB. Of these, only ten had microbiologically confirmed TB (positive Xpert and/or culture) and the remaining ten were clinically diagnosed with positive response to anti-TB treatment and were classified as unconfirmed TB. Stool Xpert had sensitivity of 100% (95%CI: 66.4-100) and specificity of 99.3% (95%CI: 96.2-100) compared to culture; however, the sensitivity was decreased to 50% (95%CI: 27.2-72.8) when compared to CRS. The Xpert on gastric aspirate had sensitivity of 77.8% (95%CI: 40-97.2) compared to culture and 40% (95%CI: 19.1-64) compared to CRS.
CONCLUSIONS: The sensitivity of Xpert for stool sample is comparable to that for gastric aspirate. Stool sample is a potential alternative to pulmonary specimen in the diagnosis of pulmonary TB in children using Xpert.

Background

Tuberculosis (TB) is one of the leading causes of death globally [1]. Children account for 11% of the global disease burden with an estimated 1.1 million new childhood TB cases and 205,000 pediatric TB deaths in 2018 [1]. Early detection of TB is critical for timely initiation of treatment but is challenging in children because of non-specific clinical presentations, difficulty of obtaining respiratory specimen and lack of sensitive diagnostic tests [2].

Sputum specimen remains being the most frequently used clinical sample to confirm the diagnosis of TB microbiologically [3]. However, in most cases, children are unable to expectorate sputum specimen and when sputum is available; the yield is expected to be poor because of the paucibacillary nature of childhood TB. As a result, specimen obtained through different procedures such as induced sputum, bronchoalveolar lavage, and gastric aspirate have been studied to improve the sensitivity of microbiological examinations [4]. Though these procedures are well tolerated in adults, they are relatively uncomfortable for children, and the capability to carry out these procedures may be lacking in many TB endemic settings. Thus, there is a need for non-sputum based specimen to diagnose TB in children who are unable to expectorate sputum.

Stool is an alternative specimen for TB diagnosis, because Mycobacterium tuberculosis (MTB) can be swallowed with the sputum and detected in stool [5]. In particular, stool is easy to obtain from infants and young children who are unable to produce sputum [6]. The introduction of Xpert has revolutionized the diagnosis of TB and the World Health Organization (WHO) has endorsed Xpert for the diagnosis of TB from various types of specimen such as sputum, lymph node tissue and aspirate, cerebrospinal fluid, gastric lavage and aspirate [7]. Recently, studies evaluating Xpert for detection of pulmonary TB (PTB) from stool have been published with highly variable sensitivities, ranging from 32% to 83.3% [6, 8]. In Ethiopia, there is a paucity of data on the diagnostic performance of Xpert on stool specimen for children who are unable to expectorate sputum. Therefore, this study is aimed to evaluate the diagnostic performance of Xpert for the diagnosis of PTB from stool specimen in children with presumptive TB.

Materials and methods

Study design and setting

An institution-based cross-sectional study was carried out among consecutively recruited children (less than 15 years old) with presumptive PTB at Jimma University Medical Center (JUMC), Ethiopia between March and November 2019. The study area is located in Jimma Zone, Oromia Regional State in Southwest part of Ethiopia. Jimma town is located 357km away from Addis Ababa (the capital city of Ethiopia). JUMC, located in Jimma town, is one of the oldest public hospitals in the country. JUMC is a teaching and referral hospital that provides services for about 15 million people in its catchment area. Each year, JUMC reports serving approximately 15, 000 inpatient hospital stays, 160, 000 outpatient visits, and 11, 000 emergency department visits [9]. Most of the patients getting services at the center are from rural area. According to WHO 2021 report, Ethiopia is among the 30 countries with high TB and TB/HIV burden, with an estimated annual incidence of 151,000 cases and an estimated 19,000 TB-related deaths (excluding HIV associated TB deaths).

Study participants

Participants were children aged less than 15 years presenting with a chronic cough of >2 weeks, weight loss, loss of appetite, persistent fever without an apparent cause, night sweats, or history of close contact with a TB patient within the preceding 12 months. They were classified as the presumptive PTB patients. Participants were excluded, if they had an already confirmed TB, had been on anti-TB treatment (ATT) for >72 hours before enrolment and clinical symptoms or physical signs suggestive of extrapulmonary TB.

Study procedures

Children up to 15 years of age were screened for TB and those presumptive TB cases were enrolled into this study. TB screening was done as per standard of care, which mainly relies on self-reporting of symptoms suggestive of TB (cough, fever, night sweats, weight loss and history of close contact with TB patients). Study subjects were diagnosed with confirmed TB based on positive microbiological test results (positive LJ culture and/or positive Xpert on gastric aspirate or sputum) or with unconfirmed TB based on a clinical diagnosis and positive response to ATT. Some of the patients with negative microbiologic results were given empirical treatment based on the evaluation by the treating clinicians and were evaluated for response to treatment (resolution of symptoms, weight gain, and radiographic improvement) after two months. Demographic data and clinical information of the participants were collected through structured questionnaires. Clinical data such as radiologic findings, clinical improvement after ATT, HIV status, nutritional status, and BCG vaccination status were obtained from the participants’ medical records.

Specimen collection and laboratory procedures

One respiratory specimen (a minimum of 4ml) and one stool specimen (a minimum of 3gm) were collected per child. Xpert was performed at JUMC Laboratory from respiratory specimens as part of the routine practice at the medical center, whereas TB culture and stool Xpert were performed at Jimma University Mycobacterium Research Center (JUMRC) for the research purposes.

The respiratory samples were collected as per the standard of care. The treating physician collected single gastric aspirate or expectorated sputum. Gastric aspirate samples were collected early in the morning through nasogastric tube following an overnight fasting and tested by Xpert and LJ culture. Expectorated sputum instead of gastric aspirate was used in the patients who were able to produce sputum.

Respiratory specimens were divided into two parts; the first part was diluted (2:1v/v) with Xpert sample reagent, followed by vigorous shaking and incubation at room temperature for 15 minutes. Then, 2ml of liquefied specimen was aspirated using sterile pipette provided with Xpert kit and added to Xpert cartridge and loaded in to the GeneXpert instrument to run the test. The results were reported to the treating physicians as soon as possible for participants’ management.

The second part of respiratory specimen was neutralized with phosphate buffered solution (PBS) immediately after collection and stored at 4–8 °C for 2–3 days until processing for culture. Equal volume of N-acetyl-L-Cysteine and 4% sodium hydroxide solution were added to the 50ml specimen tube. After mixing on the vortex, the sample tube was left standing for 20 minutes at room temperature for liquefaction and decontamination. Then, sterile PBS (pH6.8) was added up to 45ml to neutralize the alkaline solution, followed by centrifugation at 3000g for 15 minutes. After discarding the supernatant, the pellet was re-suspended in 2ml PBS and two drops of the resulting diluted deposit was inoculated on the slants of LJ medium.

Stool specimen (approximately 3-5g) was collected in a wide mouthed specimen collection jar on spot or submitted the following day and was stored at −20°C until processed as described previously [10]. Stool Xpert was performed by single step, centrifuge-free stool processing protocol adapted from KNCV TB foundation [10]. Briefly, 1 g of thawed stool was transferred to 50ml falcon tube using wooden applicator stick; 8ml of sample reagent was poured in the sample tube and mixed very well, and left undisturbed for 20 minutes at room temperature. The supernatant was carefully aspirated and added to Xpert cartridge using sterile pipette.

Diagnostic classification for analysis

Children were classified into three categories based on their clinical, radiological, and laboratory results. (i) ‘‘Confirmed TB”:–child has symptoms suggestive of TB and TB disease is confirmed microbiologically (positive gastric aspirate Xpert and/or culture); (ii) ‘‘Unconfirmed TB”:- child has at least 2 of the following: symptoms suggestive of TB, chest radiograph consistent with TB, or documented exposure to MTB and positive response to ATT but TB disease is not confirmed microbiologically (negative gastric aspirate Xpert and/or culture); (iii) ‘‘Unlikely TB”:- no criteria for “Unconfirmed TB” was met and TB is not confirmed microbiologically (negative gastric aspirate Xpert and/or culture). CRS is defined as either confirmed TB or unconfirmed TB in the definition of TB and cases that met “unlikely TB” criteria were classified as not TB.

Data analysis

Data were entered in Epidata version 3.3 and analyzed using SPSS software package version 20. The Xpert sensitivity, specificity, positive, and negative predictive values and their 95% confidence intervals (95%CI) were calculated using the following as one of the reference standards: i) LJ culture alone; ii) LJ culture and/or Xpert (confirmed TB); and iii) composite reference standard (CRS) (confirmed TB and Unconfirmed TB). Differences and similarities between the two methods (stool Xpert versus gastric aspirate Xpert) was determined based on the 95%CI. Non-over lapping 95% CIs indicated a difference between the two methods (stool Xpert versus gastric aspirate Xpert) and vice versa.

Ethical consideration

Ethical approval (Protocol number IHRPGD552/18) was obtained from Institutional Review Board of Jimma University, Ethiopia. We obtained written informed consent from parents or legal guardians of children and assent from children above ten years old.

Results

Characteristics of study participants

A total of 178 children with presumptive PTB were enrolled to the study. Out of these, 26 children were excluded from the final analysis: 17 were unable to provide stool specimen and 9 had contaminated culture results. We included the remaining 152 participants for whom we analyzed 17 expectorated sputum specimens, 135 gastric aspirates and 152 stool specimens. Seventy-eight (51.3%) of 152 participants were females and participants’ ages ranged from 7 months to 14 years (median 3 years). Majority, 98 (64.5%), of the participants were rural residents (Table 1). Regarding the clinical manifestation of the participants, 141 (92.8%) had cough for >2 weeks, 122 (80.3%) had loss of appetite, 109 (71.7%) had fever, 90 (59.2%) had weight loss, 90 (55.9%) had weakness/fatigue, 70 (46.1%) had shortness of breath and 35 (23%) had TB contact history. The majority, 142 (93.4%), were vaccinated with BCG. Close to a third of the participants, 54 (35.5%), were severely malnourished, whereas 6 (3.9%) of them were on anti-retroviral therapy.

Table 1

Demographic characteristics of participants with their diagnostic results.

Variable	Culture positive n(%)	Culture negative n(%)	GA Xpert positive n(%)	GA Xpert negative n(%)	Stool Xpert positive n(%)	Stool Xpert negative n(%)	P-value
Age (years)
< 1	1(0.7)	13(7.2)	1(0.7)	11(7.2)	1(0.7)	11(7.2)	0.7
1–4	6(3.9)	73(48)	5(3.3)	74(48.7)	7(4.6)	68(44.7)
5–10	1(0.7)	38(25)	1(0.7)	38(25)	1(0.7)	36(23.7)
11–14	1(0.7)	21(13.8)	1(0.7)	21(13.8)	1(0.7)	20(13.2)
Gender
Male	0	74(48.7)	1(0.7)	73(48)	1(0.7)	68(44.7)	0.03
Female	9(5.9)	69(45.4)	7(4.6)	71(46.7)	9(5.9)	67(44.1)
Residence
Urban	2(1.3)	52(34.2)	1(0.7)	53(34.9)	2(1.3)	49(32.2)	0.4
Rural	7(4.6)	91(59.9)	7(4.6)	91(59.9)	8(5.30	86(56.6)

GA = gastric aspirate

Diagnosis of TB

Out of the 152 pulmonary specimens analyzed, MTB was detected in 9 (6.7%) by culture and in 8 (5.9%) by Xpert. In one of the participants, gastric aspirate Xpert was positive whereas the culture from the same specimen was negative. On the other hand, in two of the participants, culture from the gastric aspirates were positive whereas Xpert results were negative. All the 17 expectorated sputum specimens were negative by both Xpert and culture. Stool Xpert testing revealed 10 (6.6%) MTB positive cases. Rifampicin resistance was not detected in any of the Xpert positive specimens. Table 2 shows results of gastric aspirate Xpert, stool Xpert and gastric aspirate culture amongst study subjects.

Table 2

Proportion of positive GA Xpert and stool Xpert compared to GA culture for MTB.

GA Xpert	GA Culture			p-value
	Positive n(%)	Negative n(%)	Total n(%)
Positive	7(4.6)	1(0.7)	8(5.3)	0.00
Negative	2(1.3)	142(93.4)	144(94.7)
Total	9(5.9)	143(94.1)	152(100)
Stool Xpert	GA Culture			p-value
	Positive n(%)	Negative n(%)	Total n(%)
Positive	9(6.2)	1(0.7)	10(6.9)	0.00
Negative	0	135(93.1)	135(93.1)
Total	9(6.2)	136(93.8)	145(100)

MTB = Mycobacterium tuberculosis, GA = gastric aspirate

Overall, 10 (6.6%) of the 152 study participants had microbiologically confirmed PTB, defined as a positive result on culture and/or Xpert on pulmonary specimen. We reviewed the medical records of the remaining 142 (93.4%) of participants who were microbiologically negative. Of these, 8.5% (12/142) were clinically diagnosed as TB by the clinicians. Among the 12 clinically diagnosed TB cases, 83.3% (10/12) showed clinical improvement after ATT. The remaining 2 cases died after two weeks of ATT initiation and were classified as unlikely TB cases. Out of the 10 cases who have improved clinically, 4 had radiological findings suggestive of TB whereas 6 had no radiological evidences of TB documented on their medical records and hence were classified as “unconfirmed TB”. However, none of the children with clinically-diagnosed TB (unconfirmed TB) had positive stool Xpert result. In the remaining 91.5% (130/142) of the cases, TB was ruled out and an alternative diagnosis was made and hence, they were classified as “unlikely TB” (Fig 1).

Fig 1

Flow chart describing study workflow and diagnostic classifications.

TB = tuberculosis, PTB = pulmonary tuberculosis, n = number, +Ve = positive, -Ve = negative, LJ = Lowenstein-Jensen.

Diagnostic performance of stool Xpert

Error/invalid results were documented in 4.6% (7/152) of Xpert tests performed on stool. Due to a shortage of Xpert cartridges, we didn’t repeat the tests in these invalid cases. Of the 145 stool specimens with valid Xpert results, 6.9% (10/145) were MTB positive. Stool Xpert detected all gastric aspirate culture confirmed TB cases and one positive case missed by gastric aspirate culture (Table 2). Moreover, stool Xpert also detected two MTB positive cases which were found to be negative on gastric aspirate Xpert.

Using culture of the respiratory specimen (gastric aspirate and expectorated samples) as the reference standard, stool Xpert had sensitivity of 100% (95% CI: 66.4–100) and specificity of 99.3% (95% CI: 96.2–100), whereas gastric aspirate Xpert had sensitivity of 77.8% (95% CI: 40–97.2) and specificity of 99.3% (95% CI: 96.2–100) (Table 3). Using gastric aspirate culture and/or Xpert positivity (microbiological confirmation) as one of the reference standards, stool Xpert had each 100% sensitivity, specificity, PPV and NPV (Table 4 and S1 Table).

Table 3

Diagnostic performance of GA Xpert and stool Xpert using culture as a reference standard.

Tests	Diagnostic performances
	Sensitivity % (95%CI)	Specificity % (95%CI)	PPV% (95%CI)	NPV % (95%CI)
GA Xpert	77.8(40–97.2)	99.3(96.2–100)	87.5(49–98.1)	98.6(95.5–99.6)
Stool Xpert	100(66.4–100)	99.3(96.2–100)	90(56.1–98.5)	100

GA = gastric aspirate, CI = confidence interval, PPV = positive predictive value, NPV = negative predictive value

Table 4

The diagnostic performances of stool Xpert and GA Xpert using LJ culture and/or GA Xpert (microbiological confirmations) as one of the reference standards.

Tests	Diagnostic performances
	Sensitivity % (95%CI)	Specificity % (95%CI)	PPV % (95%CI)	NPV % (95%CI)
Stool Xpert	100	100	100	100
GA Xpert	80(79.2–80.8)	100	100	98.6(98.6–98.7)

GA = gastric aspirate, CI = confidence interval, PPV = positive predictive value, NPV = negative predictive value

Moreover, the sensitivity, specificity, PPV, and NPV of stool and gastric aspirate Xpert were also calculated with reference to CRS. Accordingly, stool Xpert had a sensitivity of 50% (95% CI; 27.2–72.8), specificity of 100% (95% CI: 97.1–100), PPV of 100% and NPV of 92.6% (95% CI; 89–95.1) against CRS (Table 5). The corresponding sensitivity, specificity, PPV and NPV for gastric aspirate Xpert were 40% (95% CI; 19.1–64), 100% (95% CI; 97.2–100), 100% and 91.7% (95% CI; 88.5–94) respectively compared to CRS (Table 5). Stool and gastric aspirate Xpert MTB detection rates compared to CRS is also shown in supporting information (S2 Table).

Table 5

The diagnostic performance of GA Xpert, stool Xpert and GA culture compared to CRS.

Tests	Diagnostic performances
	Sensitivity % (95%CI)	Specificity % (95%CI)	PPV % (95%CI)	NPV % (95%CI)
GA Xpert	40(19.1–64)	100(97.2–100)	100	91.7(88.5–94)
Stool Xpert	50(27.2–72.8)	100(97.1–100)	100	92.6(89–95.1)
GA culture	45(23.1–68.5)	100(97.2–100)	100	92.3(89–94.7)

GA = gastric aspirate, CI = confidence interval, PPV = positive predictive value, NPV = negative predictive value, CRS = composite reference standard

Discussion

Ethiopia is among the 30 countries with high-burden of TB and TB/HIV in the world and there is a huge number of undiagnosed TB in children [11]. The national guidelines for TB care in Ethiopia recommends Xpert for the diagnosis of childhood TB. However, obtaining appropriate respiratory specimens from children is difficult. Thus, a non-invasive sample for the diagnosis of TB in children would improve care for this population. In the current study, we have demonstrated that stool Xpert has comparable performance with gastric aspirate Xpert in consecutively recruited children with presumptive PTB. Other studies have also reported promising findings [6, 12].

We found that Xpert was 100% sensitive for MTB detection in stool among children with microbiological confirmation from a respiratory specimen and 50% sensitive in children with microbiologically and/or clinically diagnosed TB. Other studies [8, 13, 14] conducted in Ethiopia, Kenya and Pakistan have also reported excellent sensitivity (ranging from 88.9% to 100%) of stool Xpert compared to gastric aspirate culture. The lower sensitivity of stool Xpert compared to CRS (microbiological and/or clinically confirmed TB) is possibly due to the presence of paucibacillary disease in patients clinically diagnosed for TB.

The specificity of stool Xpert in our study when compared to culture was consistent with other previous studies conducted in Ethiopia, South Africa and Egypt which have demonstrated specificities ranging from 99.3 to 99.7% [6, 8, 15]. The specificity of stool Xpert in our study when compared to CRS was similar with another study conducted in Pakistan [14]. One culture negative gastric aspirate specimen was found to be positive on both stool and gastric aspirate Xpert tests. This may be due to the presence of non-viable bacilli during decontamination process of gastric aspirate specimen.

In this study, stool Xpert identified all of the gastric aspirate Xpert positive cases, suggesting stool as a potential alternative for use to gastric aspirate in the routine diagnosis of PTB. Gastric aspiration may be used to retrieve pulmonary specimen from patients who cannot expectorate sputum but it is an invasive procedure and needs trained health workers. As a non-invasive sample, stool is considered to be safe, easy for collection and has the potential to be used for detection of MTB. Moreover, stool Xpert test is performed by simple stool testing protocol adapted from KNCV TB foundation [11], which omits some labor intensive steps such as homogenization, decontamination and centrifugation of stool specimen prior to Xpert testing done by other studies [13-15]. This approach significantly reduces sample processing time, minimizes the workload on the laboratory personnel and also minimizes costs.

In our study, unfortunately, none of the clinically diagnosed TB cases were detected by stool Xpert. Poor performance of stool Xpert in the clinically diagnosed (microbiologically negative) children has also been reported in other studies [15, 16]. This could potentially indicate the limitation of the stool Xpert since patients with such clinically-diagnosed unconfirmed TB are more likely to have paucibacillary disease. Children with a high clinical probability of TB despite a negative stool Xpert should be started on ATT until better and more sensitive tools are available.

Older children are able to expectorate sputum and may produce more adult-type sputum. However, in our study, none of the expectorated sputum samples were positive for M. tuberculosis. This could be due to the poor sample quality in the expectorated sputum samples. However, we did not assess the quality of the sputum sample in the current study which might be taken as a limitation.

Our study has some limitations. We used small sample size due to resource constraints. An additional limitation of our study is the fact that, we collected only single respiratory and stool specimen rather than successive specimens which could have probably increased the yield of the tests done.

Conclusion

The sensitivity of stool Xpert is comparable to that of gastric aspirate Xpert. Stool is a potential alternative to pulmonary specimen in the diagnosis of PTB in patients who cannot produce sputum. Moreover, stool collection is easier and relatively safe compared to pulmonary specimen and can be easily implemented at lowest level of health care system. However, the diagnostic yield of stool Xpert still requires further validation and optimization using larger sample size.

The stool Xpert and GA Xpert MTB detection rate compared to LJ culture and/or GA Xpert positivity (microbiological confirmation).

LJ = Lowenstein-Jensen, MTB = Mycobacterium tuberculosis, GA = gastric aspirate.

(DOCX)

Click here for additional data file.

Stool Xpert, GA Xpert and GA culture MTB detection rate compared to composite reference standard (confirmed and unconfirmed TB).

(DOCX)

Click here for additional data file.

The original SPSS dataset used and analyzed in the current study.

(SAV)

Click here for additional data file.

9 Aug 2021

PONE-D-21-15359

To: Stool-based Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children at a Teaching and Referral Hospital in Southwest, Ethiopia

PLOS ONE

Dear Dr. Tadesse,

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.

Please submit your revised manuscript by Sep 23 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see:  http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at  https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Silvia Chiang

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

2. No Funding:

"Thank you for stating the following financial disclosure:

“No”

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments/Funding Section of your manuscript:

“This work was supported by Jimma University, Institute of Health and Jimma University-Mycobacteriology Research Center, Jimma, Ethiopia. The funders had no role in study design, data acquisition, analysis and interpretation, or the decision to prepare the manuscript and submit for publication.”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text

from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“No”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b. If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

5. Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

7. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

Additional Editor Comments (if provided):

In addition to addressing the reviewer's comments below, please also address the following issues:

1. I agree with the reviewer's first two major comments and would also add that the authors should explicitly explain how they use this classification in their analysis under the "Data analysis" paragraph.

2. Abstract/Methods, line 32 and all subsequent references to the composite reference standard (CRS): I would be better to say "clinical diagnosis with improvement after anti-TB treatment" instead of just "clinical improvement after anti-TB treatment."

3. Introduction, line 65: What do you mean by "most significant clinical sample?" The "most frequently used?" Please be more specific.

4. Introduction, lines 66-70: The yield is not poor because of many young children's inability to expectorate, but it is poor because of the paucibacillary nature of most childhood TB. Children's inability to expectorate means that other methods to obtain specimens are needed.

5. Introduction, line 71: The collection of sputum is not unsafe for children. These procedures are well tolerated; they are just resource-intensive, as the authors said, and also the capability to carry out these procedures may be lacking in many TB endemic areas. Please either justify or take out this reference to the procedures being unsafe.

6. Methods: Please add a sentence about the incidence of TB in Ethiopia, and please also briefly describe the setting of the study site (e.g. is it urban or rural? are most patients publicly insured? from rural settings? etc.)

7. Methods, lines 94-96: Please specify that these participants were classified as the presumptive TB patients.

8. Methods: Could the authors specify whether a minimum volume was collected for each respiratory sample?

9. Methods, lines 165-167: Do you mean "legal guardians" instead of "caregivers" (just wondering if only parents and legal guardians are allowed to sign informed consent)? Also, what was the minimum age for obtaining assent from the child?

10. Results, line 189: Please replace ". . . not detected in all . . ." with ". . . not detected in any . . ."

11. Results and Discussion: In Methods, the authors state that one of the reference standards is positive culture; I assume this refers to positive culture from any respiratory specimen, not just gastric aspirate. I understand that none of the expectorated sputum samples were positive for M. tuberculosis. However, instead of referencing microbiological confirmation from GA as the reference standard, please reference microbiological confirmation from a respiratory specimen.

12. In addition, I find it odd that none of the expectorated sputum samples were positive for M. tuberculosis. I would expect the older kids to be able to expectorate sputum, and they tend to have more adult-type TB. Could the authors provide an explanation? Were there a lot of epithelial cells indicating poor sample quality in the expectorated sputum samples?

13. Figure 1 is good but could be improved with a few minor changes. I would take away the fist box "children visit JMC." I'd use the same term "presumptive TB cases" in the second box to be consistent w/ the rest of the manuscript. In the "results available" box I would take out the reference to the 10 that improved on ATT and instead add that to the culture negative --> Xpert negative part (so you can indicate how many were classified as not TB and how many were classified as unconfirmed TB). I would also take out the box that says "152 patients were included in the analysis" because it's repetitive.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors evaluated the performance of stool-based Xpert MTB/RIF testing for childhood TB at a referral hospital in Ethiopia using the KNCV stool processing method. The study is of importance in the field given the need for non-sputum, non-invasive approaches to childhood TB, and the manuscript is well-written. The main limitation of the paper is that there were only 10 culture-confirmed TB cases and 10 clinically-diagnosed cases, which prevents clear conclusions on the performance of stool-based testing; however, these initial findings are promising and important to share. Areas to address to improve this manuscript include:

Major

1. The TB classification used was confirmed/probable/possible/not TB; the childhood TB classification was updated in 2015 (Graham et al. CID): https://academic.oup.com/cid/article/61/suppl_3/S179/355883

Adjusting their classification to this structure (Confirmed/Unconfirmed/Unlikely) would be important to allow comparison with other studies and any meta-analyses

2. Similarly, the CRS is defined as a combining Unconfirmed TB with Confirmed TB in the definition of TB, and Unlikely TB as not TB. Again, would adjust definitions accordingly to be consistent with other studies. Would also move that up to the section on diagnostic classification

3. Would note what measures of diagnostic accuracy were used in the methods, including sensitivity, specificity, positive and negative-predictive values

4. In methods, please describe your approach to comparing were sputum and stool-based Xpert. Options include comparing 95% CIs, as well as McNemar’s paired test of proportions. Would be clear in the methods how similarity vs. difference in the two methods are defined, as that is one of your main conclusions.

5. Was smear microscopy performed on the respiratory specimens? If so, please indicate the results and stratify performance smear status

6. Was HIV testing done? Please indicate if done and HIV prevalence

7. In the 2 cases that died, how long were they taking the ATT? Would note the duration, because if soon after initiation, would exclude as unclassifiable

8. In determining the performance of stool Xpert for Confirmed cases, please use the 10 cases (9 culture confirmed, 1 GA Xpert positive) as opposed to 9 culture-positive only

Minor

1. To increase the work’s impact, would note earlier that a single step, centrifuge-free stool processing method was used earlier in the abstract/introduction, as this approach in particular is of interest in the field, including the KNCV kit.

2. There are some spacing issues, such as in line 61 “1.1million” and “in2018” in line 62. Please ensure spacing is okay throughout

3. Were invalid stool results repeated?

4. Please indicate in the results that none of the children with clinically-diagnosed TB (probable/possible) were stool Xpert positive.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

4 Nov 2021

Response to Editor and Reviewers

We appreciate the editor and reviewers for the constructive comments which we have used to improve the quality of the manuscript. We have re-written some portions of the manuscript accordingly. We have carefully addressed the comments line by line as follows.

Editor’s comments to the Author:

Response to Editor’s comments:

Comment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_ main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf .

Response 1: We have rechecked our manuscript for the PLOS ONE’s requirement and revised it accordingly.

Comment 2: Further clarification on source of funding.

Response 2: Thank you for your feedback on the source of funding. No specific funding was obtained for this research work and we have included the following statement in the revised version under the “Funding sources’’ subheading: ‘The authors received no specific funding for this work.’

Comment 3: We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text

Response 3: No funding information was presented in the acknowledgment part. Regarding comments raised on source of funding, we have addressed them above (Response 2). I feel that we don’t need to update the funding statement. The funding statement can still read as “No”.

Comment 4: We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly.

Response 4: We have uploaded our data set as a supporting information file (S1 File). We have addressed this in the revised manuscript.

Comment 5: Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option.

Response 5: As it has been indicated in the manuscript cover page, the corresponding author is affiliated to Mycobacteriology Research Center, Jimma University, Jimma, Ethiopia.

Comments 6: PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager.

Response 6: Here is the ORCID iD of the corresponding author: https://orcid.org/0000-0003-4751-2225 .

Comment 7: Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

Response 7: We have checked that ethics statement is mentioned only in the method section under the subheading “Ethical considerations”.

Additional Editor Comments (if provided):

Response to Editor’s comment

Comment 1: I agree with the reviewer's first two major comments and would also add that the authors should explicitly explain how they use this classification in their analysis under the "Data analysis" paragraph.

Response 1: We appreciate the editor and the reviewer for the interesting feedback on TB diagnostic classification. This is well accepted comment and we have adjusted the TB diagnostic classification as it was proposed by Graham et al, 2015 and described it in the methods section under the Diagnostic classification for analysis subheading.

Comment 2: Abstract/Methods, line 32 and all subsequent references to the composite reference standard (CRS): I would be better to say "clinical diagnosis with improvement after anti-TB treatment" instead of just "clinical improvement after anti-TB treatment."

Response 2: The editor’s feedback was found interesting and we have modified it accordingly.

Comment 3: Introduction, line 65: What do you mean by "most significant clinical sample?" The "most frequently used?" Please be more specific.

Response 3: We have changed the phrase to “the most frequently used’’.

Comment 4: Introduction, lines 66-70: The yield is not poor because of many young children's inability to expectorate, but it is poor because of the paucibacillary nature of most childhood TB. Children's inability to expectorate means that other methods to obtain specimens are needed.

Response 4: We partly agree with the Editor’s comment. No doubt that sputum is the most frequently utilized sample for the diagnosis of pulmonary TB. However, in most cases, children are unable to expectorate sputum specimen and when sputum is available, the yield is expected to be poor because of the paucibacillary nature of most childhood TB. This is included in the revised manuscript.

Comment 5: Introduction, line 71: The collection of sputum is not unsafe for children. These procedures are well tolerated; they are just resource-intensive, as the authors said, and also the capability to carry out these procedures may be lacking in many TB endemic areas. Please either justify or take out this reference to the procedures being unsafe.

Response 5: The feedback from the editor is found interesting and we have modified it accordingly.

Comment 6: Methods: Please add a sentence about the incidence of TB in Ethiopia, and please also briefly describe the setting of the study site (e.g. is it urban or rural? are most patients publicly insured? from rural settings? etc.)

Response 6: We have included these information accordingly in the revised version.

Comment 7: Methods, lines 94-96: Please specify that these participants were classified as the presumptive TB patients.

Response 7: We agreed with the Editor’s comment and we have specified accordingly.

Comment 8: Methods: Could the authors specify whether a minimum volume was collected for each respiratory sample?

Response 8: A minimum volume of 4ml for respiratory specimen and 3gm of stool specimen were collected. We have specified this in the revised manuscript.

Comment 9: Methods, lines 165-167: Do you mean "legal guardians" instead of "caregivers" (just wondering if only parents and legal guardians are allowed to sign informed consent)? Also, what was the minimum age for obtaining assent from the child?

Response 9: We have replaced the term “caregivers” by “legal guardians’’ in the revised manuscript. The minimum age for obtaining assent from children was 11 years age. We have indicated this in the revised manuscript.

Comment 10: Results, line 189: Please replace ". . . not detected in all . . ." with ". . . not detected in any…"

Response 10: We have replaced it accordingly.

Comment 11: Results and Discussion: In Methods, the authors state that one of the reference standards is positive culture; I assume this refers to positive culture from any respiratory specimen, not just gastric aspirate. I understand that none of the expectorated sputum samples were positive for M. tuberculosis. However, instead of referencing microbiological confirmation from GA as the reference standard, please reference microbiological confirmation from a respiratory specimen.

Response 11: The comment is well accepted and we have modified it accordingly.

Comment 12: In addition, I find it odd that none of the expectorated sputum samples were positive for M. tuberculosis. I would expect the older kids to be able to expectorate sputum, and they tend to have more adult-type TB. Could the authors provide an explanation? Were there a lot of epithelial cells indicating poor sample quality in the expectorated sputum samples?

Response 12: Thank you for the interesting and critical observation. As you rightly stated, older children could expectorate sputum and may produce more adult-type sputum. However, in the current study, none of the expectorated sputum samples was positive for MTB. This could be due to the presence of a lot of epithelial cells indicating poor sample quality in the expectorated sputum samples. However, we didn’t assess the quality of the sputum sample in the current study and we are unable to comment on it. This is addressed in the discussion section.

Comment 13: Figure 1 is good but could be improved with a few minor changes. I would take away the fist box "children visit JMC." I'd use the same term "presumptive TB cases" in the second box to be consistent w/ the rest of the manuscript. In the "results available" box I would take out the reference to the 10 that improved on ATT and instead add that to the culture

negative --> Xpert negative part (so you can indicate how many were classified as not TB and how many were classified as unconfirmed TB). I would also take out the box that says "152 patients were included in the analysis" because it's repetitive.

Response 13: We have modified the figure accordingly.

Review Comments to the Author

Response to Reviewers’ comments:

Major

Comment 1: The TB classification used was confirmed/probable/possible/not TB; the childhood TB classification was updated in 2015 (Graham et al.) adjusting their classification to this structure (Confirmed/Unconfirmed/Unlikely) would be important to allow comparison with other studies and any meta-analyses

Response 1: We are very grateful to the editor and the reviewer for the interesting feedback on TB diagnostic classification. As we have mentioned above, this is well accepted comment and we have adjusted the TB diagnostic classification as it was proposed by Graham et al, 2015 and described it in the method section under the Diagnostic classification for analysis subheading.

Comment 2: Similarly, the CRS is defined as a combining Unconfirmed TB with Confirmed TB in the definition of TB, and Unlikely TB as not TB. Again, would adjust definitions accordingly to be consistent with other studies. Would also move that up to the section on diagnostic classification

Response 2: This is also very well accepted comment from the reviewer. In the revised manuscript, this is corrected accordingly.

Comment 3: Would note what measures of diagnostic accuracy were used in the methods, including sensitivity, specificity, positive and negative-predictive values.

Response 3: We agree with the reviewer’s comment and it is indicated as follow in the revised version of the manuscript. “The Xpert sensitivity, specificity, positive, and negative predicted values and their 95% confidence interval (95%CI) were calculated compared to LJ culture and composite reference standard (CRS).’’ We calculated the Xpert sensitivity, specificity, PPV and NPV using LJ culture and CRS as reference standard, though our main conclusion comes from using CRS.

Comment 4: In methods, please describe your approach to comparing were sputum and stool-based Xpert. Options include comparing 95% CIs, as well as McNamara’s paired test of proportions. Would be clear in the methods how similarities vs. difference in the two methods are defined, as that is one of your main conclusions.

Response 4: This is also a very fascinating feedback from the reviewer. Here we would like to clarify how the diagnostic difference or similarity is determined for the two methods (stool-Xpert vs GA-Xpert) in our study. We have revised our manuscript and the following statements were included “Differences and similarities between the two methods (stool-Xpert versus GA-Xpert) is determined based on the 95%CI. Non-over lapping 95%CI dictated the presence of difference between the two methods and vice versa.’’

Comment 5: Was smear microscopy performed on the respiratory specimens? If so, please indicate the results and stratify performance smear status

Response 5: Smear microscopy was not performed in our study. We expect that the yield from smear microscopy is minimal due to the paucibacillary nature of the disease and we didn’t perform it.

Comment 6: Was HIV testing done? Please indicate if done and HIV prevalence

Response 6: HIV test was not done. We tried to retrieve HIV test result from patients’ medical records but there were lots of missed results due to poor documentation practice.

Comment 7: In the 2 cases that died, how long were they taking the ATT? Would note the duration, because if soon after initiation, would exclude as unclassifiable

Response 7: The 2 cases that died were after 2 weeks of ATT initiation. We indicated the duration in the revised version of the manuscript. We thought that if it is TB, they would have shown some clinical improvements to the ATT within 2 weeks of treatment.

Comment 8: In determining the performance of stool Xpert for Confirmed cases, please use the 10 cases (9 culture confirmed, 1 GA Xpert positive) as opposed to 9 culture-positive only

Response 8: Many studies used culture alone as a reference standard when determining the diagnostic accuracy of new or already available diagnostic tools. Culture is one of the best method to detect TB and many scholars considered culture as the gold standard method. However, for pediatric and extra-pulmonary form of TB, culture alone may not be used as a reference standard as it misses a significant number of TB cases due to the paucibacillary nature of these disease. Our main interest here is to determine the performance of stool-Xpert using culture alone as a reference standard and to compare it with the diagnostic values when CRS is used as a reference standard. However, the main conclusion of our study was derived based on Stool-Xpert performance using CRS as a reference standard.

Minor

Comment 1: To increase the work’s impact, would note earlier that a single step, centrifuge-free stool processing method was used earlier in the abstract/introduction, as this approach in particular is of interest in the field, including the KNCV kit.

Response 1: Well accepted complement from the reviewer. In the revised version, note was made on the single step, centrifuge-free stool processing method adopted from the KNCV TB Foundation.

Comment 2: There are some spacing issues, such as in line 61 “1.1million” and “in2018” in line 62. Please ensure spacing is okay throughout

Response 2: We accepted the reviewer’s comment. In the revised manuscript, this is corrected.

Comment 3: Were invalid stool results repeated?

Response 3: No; due to shortage of Xpert cartridges, we were unable to repeat invalid stool-Xpert results. We have indicated this in the manuscript.

Comment 4: Please indicate in the results that none of the children with clinically-diagnosed TB (probable/possible) were stool-Xpert positive.

Response 4: We agree with reviewer’s comment and in the revised manuscript, it was indicated as suggested by the reviewer.

Comment 6: PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Response 6: We agree to publish the peer review process.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

7 Feb 2022

6 Apr 2022

Response to Editor and Reviewers

We appreciate the editor and reviewers for the constructive comments which we have used to improve the quality of the manuscript. As usual, we have re-written some portions of the manuscript accordingly. We have also carefully addressed the comments line by line as follows.

Editor’s comments to the Author:

Response to Editor’s comments:

Journal Requirements:

Editor’s comment: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: We have checked the references list and confirmed that it is correct and complete. None of the reference cited was retracted.

Additional Editor Comments

Comment 1: English editing service of the whole manuscript.

Response 1: We are very much grateful for the editor for English editing services. We found it very helpful to improve the quality of our manuscripts.

Additional minor edits from the Editor

Comment 1: To maintain consistency with the revised definition of the composite reference standard (CRS), which now includes “confirmed TB” and “unconfirmed TB” per the updated consensus case definitions by Graham, et al. and as recommended by the reviewer, please make the following changes: Comment 1a: Abstract, lines 30-32: Diagnostic performance was calculated with reference to LJ culture and to a composite reference standard (CRS) of confirmed TB (by Xpert or culture) and unconfirmed TB (clinical diagnosis with improvement after anti-TB treatment).

Response 1a: We accepted the comment and corrected accordingly in the revised draft. Shown in Line 31-32 of cleaned version.

Comment 1b: Study procedures, lines 115:117: Study subjects were diagnosed with confirmed TB based on positive microbiological test results (positive LJ culture and/or positive Xpert on gastric aspirate or sputum), or with unconfirmed TB based on a clinical diagnosis and positive response to ATT.

Response 1b: We agree with the Editor’s suggestion and corrected accordingly. Shown in Line 116-118 of cleaned version.

Comment 2: Data analysis, lines 173-174: Please modify this sentence based on the reviewer’s comment to use culture and/or Xpert positivity as one of the reference standards.

Response 2: We found the editor’s comment important and corrected accordingly. This is shown in the data analysis (Line 175) and result sections (Table 4 and S1 Table). Line 235-237.

Comment 3: Results, lines 228-235 and relevant tables: Please modify based on the reviewer’s comment to use culture and/or Xpert positivity as one of the reference standards.

Response 3: The Editor’s/reviewer’s comment is clearly addressed in the revised version of the manuscript. This is shown in the data analysis (Line 175) and result sections (Table 4 and S1 Table). Line 235-237.

Comments 4: Background, line 60: Technically, SARS-CoV-2 has exceeded TB. I recommend saying “Tuberculosis (TB) is a global leading cause of death . . .

Response 4: It is modified as suggested by the Editor. Shown in Line 61 of cleaned version.

Comment 5: Discussion, lines 251-253: The lower sensitivity of stool Xpert compared to the CRS is because clinically diagnosed patients have paucibacillary disease. Would modify this sentence to be more precise. Also please use the abbreviation CRS to be consistent w/ the rest of the manuscript.

Response 5: We agree with the Editor’s comment and rewritten it accordingly. Look at Line 263-264 of cleaned-revised version.

Comment 6: There are minor grammatical errors throughout the manuscript. I have asked PLOS One if there is an editing service, but unfortunately, there is not. I have listed some corrections to word choice below (underlined), but I suggest the authors ask a native English speaker to review the paper before resubmission. I did not correct errors in punctuation. Comment 6a: Abstract

i. Lines 21-22: . . . challenging mainly due to the difficulty of . . .

ii. Lines 29-30: . . . after application of a single-step, centrifuge-free stool processing method . . .

iii. Line 33: . . . 152 of whom had . . .

iv. Lines 35-36: . . . only ten had microbiologically confirmed (positive Xpert and/or culture) disease, and the remaining . . .

v. Lines 37-38: . . . compared to culture; however, the sensitivity . . .

vi. Lines 41-42: . . . sensitivity for stool is comparable to the sensitivity of Xpert for gastric aspirate . . .

Comment 6b: Background

i. Lines 72-73: . . . TB endemic settings. Thus, there is a need for . . .

Comment 6c: Study design and setting

i. Lines 94-96: . . . hospitals in the country. JUMC is a teaching and referral hospital that provides services for about 15 million people in its catchment area. Each year, JUMC reports approximately 15,000 inpatient hospital stays, 160,000 outpatient visits, and 11,000 emergency department visits.

ii. Lines 98-101: . . . an estimated annual incidence of 151,000 cases and an estimated 19,000 TB-related deaths, excluding HIV-associated TB deaths.

Comment 6d: Specimen collection and laboratory procedures

i. Line 129: . . . for research purposes . . .

ii. Lines 132, 134 and all subsequent instances: Please write out “gastric aspirate” instead of using the abbreviation “GA” as this abbreviation is not commonly used and unnecessary (will be easier for readers to understand the paper without going back to look for what “GA” means)

iii. Line 138: . . . followed by vigorous shaking and incubation for 15 minutes . . .

iv. Line 145 and all subsequent instances: Please do not abbreviate “min”; please write out “minutes”

v. Line 153: . . . from KNCV TB Foundation.

Comment 6e: Diagnostic classification for analysis

i. Lines 165-166: “CRS is defined as either confirmed TB or unconfirmed TB”; cases that met “unlikely TB” criteria were not classified as TB.

Comment 6f: Data analysis

i. Lines 175-176: “Non-overlapping 95% CIs indicated a difference between the two methods (stool Xpert vs. gastric aspirate Xpert).”

Comment 6g: Results

i. Line 189: . . . stool specimens.”

ii. Lines 189-191: Seventy-eight (51.3%) of 152 participants were females, and participants’ ages ranged from 7 months to

iii. 14 years (median 3 years). [Provide number here] (64.5%) of the participants were rural residents (Table 1).”

iv. Lines 192-197: Please report the number first, followed by the percentage in parentheses.

v. Line 195: “The majority of children . . .”

vi. Line 196: “Close to a third . . .”

Comment 6h: Diagnosis of TB

i. Lines 200-206 and throughout the manuscript: Please be consistent and use either “M. tuberculosis” or “MTB”

ii. Line 208: I don’t believe PTB was defined previously. Please write out “pulmonary TB” if it was not.

iii. Lines 209-210: We reviewed the medical records of the remaining 142/152 (93.4%) of participants who were microbiologically negative.

Comment 6i: Diagnostic performance of stool Xpert

i. Line 222: . . . a shortage of Xpert cartridges, we did not repeat . . .

Comment 6j. Discussion

ii. Line 239: The national guidelines for TB care . . .

iii. Line 241: respiratory specimens . . .

iv. Line 242: . . . for this population.

v. Line 249: microbiologically confirmed and/or clinically diagnosed TB.

vi. Line 283: . . . we did not assess the quality . . .

Response 6 (6a-6j): We are very grateful to the editor for his/her time and for correcting grammatical errors throughout the manuscript. The feedback from the editor is well accepted and considered in the revised version of the manuscript. We also shared our last version of the manuscript with language expertise and they made substantial English Language editions.

Review #1 Comments to the Author

Response to Reviewers’ comments:

Comment 1: I thank the authors for their thoughtful review of the comments, and have addressed the majority of them. For response 8, as the authors note culture alone will miss pediatric cases due to their paucibacillary disease. There are two goals of this analysis, one to provide an estimate of stool Xpert performance and the other to compare to respiratory specimen testing. In the first goal, the standard Confirmed TB definition should be used that includes Xpert results. For the second goal, it is reasonable to only use culture to allow comparison between the Xpert stool and GA to reduce bias. Would recommend presenting the results in these two ways; based on my understanding of the tables, this should still lead to 100% sensitivity and support their discussion line that all Xpert GA results were stool Xpert positive. Excluding Xpert from the main estimation results also confuses the comparison from microbiological to composite reference standard, as the CRS includes both culture and Xpert positive cases.

Response 1: We found that the feedback from the reviewer #1 is very interesting and the comment is addressed in method and results sections. In S1 Table (supporting information), stool Xpert and GA Xpert MTB detection rate compared to LJ culture and/or GA Xpert positivity (microbiological confirmation) was described. Moreover, the diagnostic performances of stool Xpert and GA Xpert using LJ culture and/or GA Xpert (microbiological confirmations) as one of the reference standards was indicated in Table 4. This is shown in the data analysis (Line 175) and result sections (Table 4 and S1 Table). Line 235-237.

Comment 2: 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Response 2: We agree to publish the peer review process.

Comment 3: While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Response 3: We have checked our figure by PACE and found that it met PLOSONE requirements.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

13 Apr 2022

Stool-based Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children at a teaching and referral hospital in Southwest Ethiopia

PONE-D-21-15359R2

Dear Dr. Tadesse,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Silvia S. Chiang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

26 Apr 2022

PONE-D-21-15359R2

Stool-based Xpert MTB/RIF assay for the diagnosis of pulmonary tuberculosis in children at a teaching and referral hospital in Southwest Ethiopia

Dear Dr. Tadesse:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Silvia S. Chiang

Academic Editor

PLOS ONE