Journal Pre-proof

Stool-based Xpert MTB/RIF Ultra Assay as a tool for detecting

pulmonary tuberculosis in children with abnormal chest imaging: a
prospective cohort study

Xu-hui Liu , Lu Xia , Bin Song , Heng Wang , Xue-qin Qian ,

Jian-hao Wei , Tao Li , Xiu-hong Xi , Yuan-lin Song , Shan-qun Li ,
Douglas B. Lowrie , Xiao-yong Fan , Shui-hua Lu

PII: S0163-4453(20)30727-1
DOI: https://doi.org/10.1016/j.jinf.2020.10.036
Reference: YJINF 4936

To appear in: Journal of Infection

Accepted date: 3 October 2020

Please cite this article as: Xu-hui Liu , Lu Xia , Bin Song , Heng Wang , Xue-qin Qian ,
Jian-hao Wei , Tao Li , Xiu-hong Xi , Yuan-lin Song , Shan-qun Li , Douglas B. Lowrie ,
Xiao-yong Fan , Shui-hua Lu , Stool-based Xpert MTB/RIF Ultra Assay as a tool for detecting
pulmonary tuberculosis in children with abnormal chest imaging: a prospective cohort study, Journal
of Infection (2020), doi: https://doi.org/10.1016/j.jinf.2020.10.036

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier Ltd on behalf of The British Infection Association.

Highlights

 Stool-based Xpert MTB/RIF Ultra test showed substantial

agreement with respiratory tract sample test for paediatric

pulmonary tuberculosis.

 This test may take the place of respiratory tests in

resource-limited context, particularly in young children not

able to expectorate.

 This study provides clinical evidence of the use of this tool

based on a head-to-head diagnostic accuracy comparison

1
Original Article

Stool-based Xpert MTB/RIF Ultra Assay as a tool for detecting

pulmonary tuberculosis in children with abnormal chest

imaging: a prospective cohort study

Xu-hui Liu1,2, Lu Xia1, Bin Song3, Heng Wang4, Xue-qin Qian1, Jian-hao Wei1, Tao
Li1, Xiu-hong Xi1, Yuan-lin Song2, Shan-qun Li2, Douglas B. Lowrie1, Xiao-yong
Fan1,5,6, Shui-hua Lu5,6

1 Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China
2 Shanghai Zhongshan Hospital, Fudan University, Shanghai, China
3 Wuhan Jinyintan Hospital, Wuhan, China
4 Guiyang Pulmonary Hospital, Guiyang, China
5 Wenzhou Medical University, Wenzhou, China
6 TB Center, Shanghai Emerging and Re-emerging Institute, Shanghai, China

Correspondence:

lushuihua66@126.com (S.-H. Lu).

2901, Caolang Rd, Jinshan, Shanghai, China.

Zipcode : 201508 Phone : +86 021 37990333

OR xyfan008@126.com （X.-Y. Fan）

2
Abstract:

Objectives: To evaluate the diagnostic efficacy of stool-based Xpert MTB/RIF

Ultra assay versus other assays for the detection of paediatric pulmonary
tuberculosis (PTB).

Methods: A prospective head-to-head comparative study was conducted from

Dec 2017 to May 2019 in Shanghai Public Health Clinical Center. Samples were
collected from children (< 15 years) with abnormal chest imaging (X-ray or CT
scan) results for the following tests: Ultra on stool sample (Ultra-Stool), Ultra on
respiratory tract sample (Ultra-RTS), Xpert MTB/RIF assay (Xpert) on RTS
(Xpert-RTS), acid-fast bacilli smear on RTS (AFB-RTS), and Mycobacterium
tuberculosis (Mtb) culture on RTS (Culture-RTS). The results were compared with
a composite reference standard.

Results: A total of 126 cases with paired results were analysed. Against a
composite reference standard, Ultra-RTS demonstrated the highest sensitivity
(52%) and specificity (100%). Ultra-Stool showed 84.1% concordance with
Ultra-RTS, demonstrating 45.5% sensitivity and 94.7% specificity (kappa = 0.65,
95% CI= 0.51-0.79). The sensitivity of Ultra-Stool was similar to Mtb culture
(45.5%, p = 1.000) and higher than AFB-RTS (27.3%, p < 0.05). Assay positivity
was associated with age and infiltration range in chest imaging.

Conclusions: When RTS is difficult to obtain, stool sample-based Ultra is a

comparable alternative.

Keywords: Xpert MTB/RIF Ultra; tuberculosis; children; trace-positive; stool

3
Introduction

Case notification of paediatric pulmonary tuberculosis (PTB) is hard. The

main reason for this is the lack of child-appropriate diagnostic tools.1,2 Sputum
from young children, especially from those under five years old, is not always
available, primarily because the child often lacks the strength to expectorate. 2-5
Invasive procedures, such as gastric aspiration (GA), induced sputum (IS),
nasopharyngeal aspiration (NA), or bronchial lavage, must usually be performed
to obtain high-quality respiratory tract samples (RTS), which is difficult in
resource-limited contexts.2,3 Furthermore, the diagnostic accuracy of routine
decision-making methods, such as acid-fast bacilli (AFB) smear, Mycobacterium
tuberculosis (Mtb) culture, or nucleic amplification assay (such as Xpert
MTB/RIF), is much lower for young children than for adults, with a sensitivity of
10-30% against clinical reference standards.3,6,7

The Xpert MTB/RIF assay (Xpert; Cepheid Inc., Sunnyvale, CA, USA) is a rapid
molecular detection method for TB with high sensitivity and specificity for
culture-positive specimens, but demonstrates less sensitivity for paucibacillary
specimens, such as those from culture/smear-negative or paediatric patients.
Xpert MTB/RIF Ultra (Ultra; Cepheid Inc.), the next-generation Xpert assay, was
designed to resolve this disadvantage with reportedly outstanding
improvement.8-11 GA is considered the gold standard for obtaining RTS to
diagnose paediatric PTB because it allows sputum swallowed by children to be
collected.2, 3 However, swallowed sputum will also pass through the intestines,
making it possible to detect respiratory tract bacteria in stool samples.5,12 We
hypothesized that this ultra-sensitive assay would improve the efficacy of
utilizing children's stool samples in which Mtb from swallowed RTS can be
detected. With sufficient diagnostic accuracy, this method may be adopted as a
non-invasive, simple, and rapid diagnostic tool for paediatric PTB. We designed
this comparative accuracy study to verify the diagnostic accuracy of stool
4
sample-based Ultra.

Methods

Study design and population. A prospective cohort study was conducted to

evaluate the diagnostic performance of Xpert MTB/RIF Ultra in RTS and stool
samples at the Shanghai Public Health Clinical Center. Study participants with
suspected TB met the following criteria: 1) age < 15 years old; 2) pulmonary
lesions in chest imaging (X-ray or CT scan) including inflammatory infiltration,
nodules, cavities, and mediastinal lymphadenopathy, irrespective of close TB
exposure history or immunologic evidence (tuberculin skin test or interferon
gamma release assay); 3) willing to provide a stool sample for the purposes of
the study; 4) routine testing of RTS, including Xpert, culture, or AFB smear, was
deemed appropriate. Considering the potential trauma involved in obtaining an
extra RTS and the bias introduced from anti-TB drug exposure, we excluded
those patients with a definite diagnosis of TB before admission. Other exclusion
criteria included: 1) inadequate or invalid sample for the paralleled assays; 2)
incomplete clinical data or indeterminate clinical diagnosis; 3) history of anti-TB
therapy.

Head-to-head diagnostic accuracy comparisons between the AFB smear test

on RTS (AFB-RTS), Mtb culture on RTS (Culture-RTS), Xpert on RTS (Xpert-RTS),
Ultra on RTS (Ultra-RTS), and Ultra on stool (Ultra-Stool) were conducted.
Demographic information, clinical profile, laboratory tests, and chest imaging
results were collected during enrolment and reviewed at the endpoint.
Diagnostic performance was evaluated against the gold standard and composite
reference standard (CRS). The gold standard defining PTB in this study was a
positive culture result for Mtb obtained from RTS. The composite reference
standard (CRS) was defined in general concordance with diagnostic consensus
13-15: 1) “confirmed TB” with at least one positive result from decision-making
tests such as Mtb culture, AFB smear, FDA-approved nucleic amplification assay
5
(such as Xpert or loop-mediated isothermal amplification, but excluding Ultra to
avoid inclusion bias), histopathological examination, and sequencing method; 2)
“unconfirmed TB” with bacteriological confirmation not obtained AND other
pulmonary diseases (including pneumonia, lung tumour, lung cyst, and
interstitial lung diseases) excluded, AND at least 2 of the following: a) symptoms
suggestive of TB: fever or cough for more than 2 weeks, weight loss, or failure to
gain weight in the previous 3 months, b) household TB contact within the
preceding 24 months who had either a positive (≥ 10 mm) tuberculin skin test or
a positive IGRA assay, c) suggestive imaging evidence, d) appropriate response to
anti-TB therapy during follow-up; 3) “not TB” without bacteriological
confirmation AND criteria for “unconfirmed TB” not met during a 3-6 months
follow-up. Both “confirmed TB” and “unconfirmed TB” were considered a “TB
case” for the purposes of this study. If the final diagnosis was not established by
clinical assessment, we conducted a 3-6 months follow-up by telephone
interview or revisited to obtain diagnosis.

This study was reviewed and approved by the ethics committee of the
Shanghai Public Health Clinical Center (2018-S037-02). Written informed
consent was obtained from parental guardians of the recruited participants.

Study procedures. The first available RTS and stool samples were collected and
properly stored. All samples were processed for microbiological analysis by the
Clinical Microbiology Department of Shanghai Public Health Clinical Center. RTS,
which could be sputum (SP), or obtained by NA or GA, was tested using Xpert,
Ultra, AFB smear (Ziehl-Neelsen stain), Mtb culture (BACTEC MGIT 960 system,
BD Biosciences, Franklin Lakes, NJ), and a phenotypic drug susceptibility test for
rifampin (conducted in LJ medium). Stool samples were only tested using Ultra. If
multiple samples were tested from the same participant, the first eligible sample
result was reported in this study. Samples were generally tested within 8 h after
collection. When this was not possible, samples were frozen at -80 ℃ until use.

6
RTS was divided for use among the various tests, using an optimum volume of 5
mL for each test and a minimum volume of 1 mL.

Ziehl-Neelsen staining was performed using a kit approved by the China

Food and Drug Administration. The Bactec MGIT 960 system, Xpert, and Ultra
assay were utilized for mycobacterium detection on RTS according to the
manufacturers’ recommendations. The stool sample process procedure was
similar to that previously described.12,19 In brief, 0.30 ± 0.15 g of stool sample
was homogenized and suspended in 2-3 mL of sterile phosphate-buffered saline
solution by manual shaking, stored at 4 °C for 24 – 48 h, and then processed by

filter screen (0.5×0.5mm). The collected filtrate was mixed with 5 mL of sample

reagent, briefly agitated manually, and 2.5 mL of supernatant was transferred to

the cartridge for Ultra testing after 15 min.

Data analysis and statistical methods. Researchers responsible for data

analysis were blinded. Population size was estimated using a kappa test for
agreement between two raters on the basis of our preliminary study. The kappa
value between results of Ultra-RTS and Ultra-Stool was calculated as 0.8 in
preliminary tests, indicating substantial agreement.20 Assuming a prevalence of
70% and 25% drop out, we determined that a population size of 269 cases was
sufficient to determine agreement based on a two-sided test with = 0.1 and 80%
power. The sensitivity, specificity, positive predictive value (PPV), and negative
predictive value (NPV) of each assay were calculated. The statistical significance
of differences in positivity rate between diagnostic assays was compared by
McNemar’s test (paired) or Fisher’s exact test (unpaired). Binominal categorical
variables were compared by Chi-square test. A value of p < 0.05 was considered
statistically significant. Confidence intervals (95% CI) were estimated for
binomial distributions. Statistical analyses were performed using IBM SPSS
Statistics version 22 (IBM Corp, Armonk, NY, USA), GraphPad Prism version 5
(GraphPad Software, La Jolla, CA, USA), and PASS version 11 (NCSS, LLC. Kaysville,
7
Utah, USA).

Results:

Study participants. From Dec 2017 to May 2019, 311 cases with suspicion of
PTB were recruited for this study, of which 112 cases were excluded: 22 cases
required an extra RTS test, 19 declined to participate, 44 had inadequate samples,
and 27 had incomplete clinical profiles or indeterminate diagnoses. Consequently,
199 cases were analysed. Among them, 126 had paired results from Culture-RTS,
AFB-RTS, Xpert-RTS, Ultra-RTS, and Ultra-Stool assays. The mean participant age
was 4.15 ± 4.16 years, 70.6% (89/126) of cases were < 5 years old, and 73.0%
(92/126) of cases were inpatients. None of the cases were HIV-positive (Table 1,
Figure 1). 89.9% (80/89) of RTS from cases ≤5 years old were obtained by GA.

Comparative diagnostic accuracy of different assays for the detection of

paediatric pulmonary tuberculosis. Against CRS, Ultra-RTS performed best
with an overall sensitivity of 52.3%, which was higher than Ultra-Stool (45.5%).
The specificity of all tests was high, ranging from 94.7% to 100% (Table 2, Figure
2). A similar trend was shown against the gold culture standard (Table 3, Figure
2). The overall concordance between Ultra-Stool and Ultra-RTS was 84.1%
(106/126), kappa = 0.65 (95% CI = 0.51-0.79). Compared with routine
laboratory tests, the diagnostic accuracy of Ultra-Stool was similar to that of
Culture-RTS and Xpert-RTS assays, demonstrating moderate sensitivity and high
specificity. Lower test sensitivity was demonstrated in cases <5 years old. (Figure
2, Table S1).

Among the 88 TB cases (“confirmed TB” + “unconfirmed TB”), assay positivity

was significantly higher in cases with multilobular versus non-multilobular
infiltrates (Table S2).

Trace-positive results. The semiquantitative scale for Ultra results was trace,

8
very low, low, medium, and high; while the scale for Xpert results was very low,
low, medium, and high. Inclusion of the trace-positive category can increase
sensitivity, but potentially reduce specificity.11 In this study, the trace-positive
rate was high, contributing to 21.7% (10/46) of positive results from Ultra on
RTS samples and 38.1% (16/42) on stool samples (p =0.093). Stratified analysis
revealed stool trace-positive results were associated with a lower rate of
multilobular infiltrates in chest imaging. Trace-positivity presented in 22.2%
(12/54) of non-multilobular and 11.8% (4/34) of multilobular infiltrate cases (p
=0.266) for stool, while the rates were 14.8% (8/54) and 0.06% (2/34),
respectively (p =0.199), for RTS.

False positive from Ultra-Stool assay. Two “false positives” were obtained by
the Ultra-Stool test. The first came from an extrapulmonary TB patient
(diagnosed with intestinal and celiac TB by histopathological examination) who
also had pneumonia and received antibiotic treatment; the lung lesions
disappeared in 2 weeks. The second case was Ultra “trace-positive” on the first
stool sample and negative on the second; intestinal and lung lesions progressed
after anti-TB treatment, but subsided after glucocorticoid treatment, and
inflammatory bowel disease (IBD) was diagnosed based on histopathological
examination and clinical assessment. Both cases were negative by Culture-RTS.

Added positive yield from combined assays. We evaluated the combined

diagnostic accuracy between Ultra-RTS, Ultra-Stool, Culture-RTS, and AFB-RTS
and interpreted the results with a Venn diagram18 in clinically diagnosed cases
(confirmed and unconfirmed). The overall positivity from the four assays with
one sample was 72.7% (64/88) (Figure S1) .

Rifampin resistance detection. Indeterminate results for rifampin resistance

were commonly observed for paediatric samples: 13.0% (6/46) in Ultra-RTS,
19.0% (8/42) in Ultra-Stool, and 12.5% (4/32) in Xpert-RTS. All positive results
were consistent with the drug resistant phenotype except for one, which was
9
confirmed by Sanger sequencing for rpoB gene (Table S3).

Discussion

A rapid, high-sensitivity, decision-making, point-of-care test for paediatric PTB is

urgently needed. Unfortunately, no such solution has been previously validated.
Through this study, we validated a promising solution to these drawbacks. The
Ultra-Stool assay demonstrated suboptimal accuracy with substantial
consistency compared with the Ultra-RTS assay in HIV negative children.
Compared with routine laboratory tests, the diagnostic accuracy of the
Ultra-Stool assay was similar to that of Culture-RTS and Xpert-RTS assays, with
moderate sensitivity, short turn-around time, and the further advantage that it is
a non-invasive procedure.

Some studies have reported the diagnostic accuracy of the Ultra assay for
detection of paediatric PTB on RTS, but inconsistent sensitivity9,10,16,17. A
systematic review27 showed a pooled sensitivity of 73% from Ultra on RTS
against culture standard. This is similar to our study. Zar et al.17 presented the
value of Ultra in hospitalized children suspected of PTB (16.4% HIV positive) on
IS and NA samples. The sensitivities of a single Ultra test from NA and IS were
45.7% (16/35) and 74.3% (26/35), respectively, compared to sputum culture. 17
Sabi et al.9 evaluated the diagnostic performance of Ultra on sputum in children
suspected of PTB (52% HIV positive) from two sites. The test’s sensitivity was
64.3% with the categories “culture confirmed” and “not TB” as the reference
standard. HIV status was reportedly associated with Ultra assay positivity on
sputum.9 However, no HIV-infected participants were enrolled in this study
because HIV prevalence in children in Eastern China is extremely low. We
included children suspected of PTB with abnormal chest imaging results, which
may have potentially increased the number of positive results from tests.
Assay sensitivity was relatively lower for children < 5 years old (Table 1),

10
which was consistent with published studies. In this study, Ultra-RTS
demonstrated the highest sensitivity (47.5%) and specificity (100%) for a single
test. Ultra-Stool was suboptimal to Ultra-RTS, but comparable to Culture-RTS. In
hospitalized patients, GA is the preferred approach for obtaining RTS from young
children since GA is a stable technique for collecting qualified samples. 3,21,22
However, in resource-limited contexts, such as outpatient services, or when
screening a large population, a stool-based test would be preferable since
samples would be more readily obtainable.

In May 2020, Kabir et al.23 first published a cross-sectional study that assessed
the use of the Ultra assay on stool in the diagnosis of pulmonary tuberculosis in
children. “Bacteriologically confirmed on induced sputum” was used as the
reference standard. Of the 447 participants in that study, 29 (6.5%) were
“bacteriologically confirmed on induced sputum”, and the sensitivity and
specificity of Xpert Ultra on stool was reported as 58.6% (95% CI, 40.7-74.5) and
88.1% (86.4-92.3), respectively. A limitation of that study is the small number of
confirmed cases, which reduced the statistic power. Due to a substantial gap in
bacteriological confirmations, most studies of diagnostic assays for paediatric
PTB may be similarly challenged. In the systematic review’s report 27, studies
that evaluated Ultra assay on stool specimens against culture or a composite
reference standard were not identified. The pooled sensitivity and specificity of
Xpert on stool were 61.5% and 98.5% against culture, and 16.3% and 99.7%
against a composite reference standard.

Reported accuracy indices, such as sensitivity and specificity, may vary due to
confounding factors such as patient disease severity (associated with bacillary
concentrations in test samples), anti-TB drug exposure (including macrolides,
aminoglycosides, and carbapenems), sample volume, processing procedure, or
others. These factors are not likely to be uniformly present in studies. Thus, with
strictly paired analysis, the comparative advantages and concordance between

11
assays should be viewed as a good index to evaluate the diagnostic potential of a
novel test.

The trace-positive rate of the Ultra assay is reportedly low for adults but is
common for children.9,10,11,24 The high trace-positive rate and indeterminate
rifampin resistance detection in the current study were most likely due to lower
bacillary concentrations present in the specimens. A lower threshold may result
in higher sensitivity and is less likely to generate false-positive results if the
detection system is not contaminated. Ultra is a cartridge-based, closed detection
system, thus theoretically a “trace” score will increase the likelihood of Mtb
notification, especially for paucibacillary samples. In this study, trace positivity
comprised 21.7% of positive results with Ultra-RTS, and no false positives were
observed. The contribution was even higher for Ultra-Stool at 38.1%, but two
“false positives” were observed. The first “false positive” was from a case
diagnosed with intestinal and celiac TB plus pneumonia. Strictly speaking, this
test result was not incorrect, but simply the wrong location specification. The
second “false positive” was from a patient diagnosed with IBD, in which an
Mtb-induced inflammatory reaction was one possible cause without
development of TB.25,26 This case indicated that a trace amount of transiently
passing Mtb may result in a “false-positive”, especially in settings with high TB
prevalence. Thus, a single trace-positive result from a stool sample should be
regarded with suspicion and an additional RTS test performed if appropriate.

This study has additional limitations. First, the number of recruited

participants was limited, and even fewer were eligible for final analysis. Of the
270 children from whom samples (RTS and stool) were supposed to be collected,
only 126 could finally be included in the analysis. Therefore, the results did not
provide the presupposed statistical power. However, considering the scarcity of
young children’s data, we still believe these study findings should be very helpful.
Second, the enrolled participants were mostly inpatients because GA is not

12
usually available at outpatient services. Owing to the fact that hospital admission
standards vary, we could not precisely calculate and correct any bias resulting
from different selection criteria. This means the results do not necessarily reflect
community-based diagnostic performances.

In conclusion, our study shows that stool sample-based Ultra is a comparable

alternative to RTS based test. Considering that most unidentified paediatric PTB
patients live in resource-limited areas where appropriate respiratory samples
are difficult to obtain, testing a stool sample with Xpert MTB/RIF Ultra may
provide a useful adjunctive tool for detecting paediatric PTB. Although the
accuracy of this test alone is suboptimal, but not significantly lower than
standard tools, and because the number of unidentified paediatric TB cases is
immense contributing to the neglecting of paediatric TB, any incremental
improvement in microbiological confirmation may benefit thousands of children.

Transparency Declaration: We affirm that this manuscript is an honest,

accurate, and transparent account of the study being reported; that no important
aspects of the study have been omitted; and that any discrepancies from the
study as planned have been explained.

Conflict of Interest: Dr. Lu has nothing to disclose.

Funding: This work was supported by Grants from Chinese National Mega
Science and Technology Program on Infectious Diseases (2018ZX10302301,
2018ZX1073130), National Key R&D Program of China (2018YFC1313600), and
National Science Foundation of China (81900005, 81770011).

13
Acknowledgements: We thank all study participants, without whom this work
would not have been accomplished. We also thank Professor Qian Gao and Yao
Zhang for methodological support.

Contributions: X.-H Liu and L. Xia contributed equally to conducting the study
and writing the original draft; X.-Y. Fan and S.-H. Lu contributed equally in
conceptualization, methodology, and reviewing the original draft; X.-H Liu, L. Xia,
H. Wang, B. Song, X.Q. Qian and J. H. Wei contributed to data curation,
investigation, and validation; Y.-L. Song, S.-Q. Li and D.-B. Lowrie provided editing
and data analysis.

14
References

1. WHO. Global tuberculosis report 2019. WHO/CDS/TB/2019.15. World Health

Organization 2019.

2. WHO. Best practices in child and adolescent tuberculosis care.

WHO/CDS/TB/2018.23. World Health Organization 2018.

3. Lewinsohn D.M., et al. Official American Thoracic Society/Infectious Diseases

Society of America/Centers for Disease Control and Prevention clinical
practice guidelines: diagnosis of tuberculosis in adults and children. Clinical
Infectious Diseases 2017; 64: 111-115.

4. Moore DP., et.al. The Incremental Value of Repeated Induced Sputum and
Gastric Aspirate Samples for the Diagnosis of Pulmonary Tuberculosis in
Young Children With Acute Community-Acquired Pneumonia. Clin Infect Dis.
2017 Jun 15;64(suppl_3): S309-S316.

5. LaCourse SM., et.al. Stool Xpert MTB/RIF and urine lipoarabinomannan for
the diagnosis of tuberculosis in hospitalized HIV-infected children. AIDS.
2018 Jan 2;32(1):69-78.

6. Hatherill M, Hawkridge T, Zar HJ, et al. Induced sputum or gastric lavage for
community-based diagnosis of childhood pulmonary tuberculosis? Arch Dis
Child 2009; 94:195–201.

7. Abadco DL, Steiner P. Gastric lavage is better than bronchoalveolar lavage for
isolation of Mycobacterium tuberculosis in childhood pulmonary tuberculosis.
Pediatr Infect Dis J 1992; 11:735–8.

8. Bahr N.C., Nuwagira E., Evans E.E., et al. Diagnostic accuracy of Xpert
MTB/RIF Ultra for tuberculous meningitis in HIV-infected adults: a
prospective cohort study. Lancet Infect Dis 2018; 1: 68–75.

9. Sabi I., Rachow A., Mapamba D., et al. Xpert MTB/RIF Ultra assay for the
diagnosis of pulmonary tuberculosis in children: a multicentre comparative
accuracy study. Journal of Infection 2018; 77: 321–327.

10. Nicol M.P., Workman L., Prins M., et al. Accuracy of Xpert MTB/RIF ultra for
the diagnosis of pulmonary tuberculosis in children. Pediatr Infect Dis J 2018;
37(10): e261-e263.

11. Dorman S.E., Schumacher S.G., Alland D., et al. Xpert MTB/RIF Ultra for
detection of Mycobacterium tuberculosis and rifampicin resistance: a
prospective multicentre diagnostic accuracy study. Lancet Infect Dis 2018; 1:
76–84.
15
12. Walters E., van der Zalm M.M., Palmer M., et al. Xpert MTB/RIF on stool is
useful for the rapid diagnosis of tuberculosis in young children with severe
pulmonary disease. Pediatr Infect Dis J 2017;36(9): 837–843.

13. Stephen M. Graham., et.al. Clinical Case Definitions for Classification of

Intrathoracic Tuberculosis in Children: An Update. Clinical Infectious
Diseases. 2015;61(S3): S179–87.

14. Dunn J.J., Starke, J.R., Revell P.A. Laboratory Diagnosis of Mycobacterium
tuberculosis Infection and Disease in Children. J Clin Microbiol. 2016; 54(6):
1434–1441.

15. Berti E., Galli, L., Venturini E., et al. Tuberculosis in childhood: a systematic
review of national and international guidelines. BMC Infect Dis. 2014;
14(Suppl 1): S3.

16. Atherton RR., et. al. Xpert MTB/RIF Ultra for Tuberculosis Testing in Children:
A Mini-Review and Commentary. Front Pediatr. 2019 Feb 28;7:34.

17. Zar HJ., et. al. Tuberculosis Diagnosis in Children Using Xpert Ultra on
Different Respiratory Specimens. Am J Respir Crit Care Med. 2019 Dec
15;200(12):1531-1538.

18. Oliveros, J.C. (2007-2015) Venny. An interactive tool for comparing lists with
Venn's diagrams. http://bioinfogp.cnb.csic.es/tools/venny/index.html.

19. Chipinduro M., Mateveke K., Makamure B., et al. Stool Xpert® MTB/RIF test
for the diagnosis of childhood pulmonary tuberculosis at primary clinics in
Zimbabwe. Int J Tuberc Lung Dis 2017; 21(2): 161–166.

20. J. Richard Landis, Gary G. Koch. The measurement of observer agreement for
categorical data. International Biometric Society 1977;33(1):159-174.

21. Hatherill M, Hawkridge T, Zar HJ, et al. Induced sputum or gastric lavage for
community-based diagnosis of childhood pulmonary tuberculosis? Arch Dis
Child 2009; 94:195–201.

22. Abadco DL, Steiner P. Gastric lavage is better than bronchoalveolar lavage for
isolation of Mycobacterium tuberculosis in childhood pulmonary tuberculosis.
Pediatr Infect Dis J 1992; 11:735–8.

23. Senjuti Kabir., et al. Xpert Ultra assay on stool to diagnose pulmonary
tuberculosis in children. Clin Infect Dis. 2020 May 18;ciaa583.
doi:10.1093/cid/ciaa583.

16
24.Mishra H., et. al. Xpert MTB/RIF Ultra and Xpert MTB/RIF for diagnosis of
tuberculosis in an HIV-endemic setting with a high burden of previous
tuberculosis: a two-cohort diagnostic accuracy study. Lancet Respir Med.
2020 Feb 14. pii: S2213-2600(19)30370-4.

25. Cohn H.M., Dave M., Loftus Jr E.V. Understanding the cautions and
contraindications of immunomodulator and biologic therapies for use in
inflammatory bowel disease. Inflamm Bowel Dis. 2017; 23(8): 1301–1315.

26. Chai Q.Y., Zhang Y., Liu C.H. Mycobacterium tuberculosis: An adaptable
pathogen associated with multiple human diseases. Front Cell Infect
Microbiol. 2018; 8: 158.

27. Kay AW., et al. Xpert MTB/RIF and Xpert MTB/RIF Ultra assays for active
tuberculosis and rifampicin resistance in children. Cochrane Database Syst
Rev. 2020 Aug 27;8:CD013359.

17
Figure 1. Flow chart of participant recruitment. Unpaired results refer to any
one of the five tests being in error, not performed, or contaminated, including
Xpert-RTS, Ultra-RTS, Ultra-Stool, AFB-RTS, and Culture-RTS. RTS, respiratory
tract sample; TB, tuberculosis; AFB, acid-fast bacilli. Overall, RTS and stool
sample were obtained from 83.7% (226/27) and 100% participants, respectively.
* Of these 44 cases, 15 did not provide a sample due to incompliance or upper
respiratory tract mucosa bleeding; 9 didn’t provide sufficient sample due to the
lack of proficiency; 18 were invalid due to contamination of sample by food or
hemorrhage, or had a dried sample; and 2 had a sample missing.

18
Figure 2. Sensitivity of different test combinations for patients aged 0-15
and < 5 years. Symbols and the upper/lower limits represent sensitivity and 95%
confidence intervals. Sensitivity was assessed against composite reference
standard (a) and Mtb culture (b). RTS, respiratory tract sample; AFB, acid-fast
bacilli.

19
Table 1. Characteristics of study participants

TB Not TB All
Categorize
Confirmed Unconfirmed
All cases
n. (%) 67 (53.2%) 21 (16.7%) 38 (30.2%) 126 (100%)
Male/Female 1.23 1.62 1.24 1.29
Co-morbidities
HIV positive (%) 0 0 0 0
Other immunodeficiency (%) 0 1 (4.8%) 4 (10.5%) 5 (4.0%)
Other disease (%) 4 (6.0%) * 2 (9.5%) ** - -
Culture positive (%) 40 (59.7%) 0 (0) 0 (0) 40 (31.7%)

Ultra-RTS positive (%) 43 (64.2%) 3 (14.3%） 0 (0) 46 (36.5%）

Ultra-Stool positive （%） 40 (59.7%) 0 (0) 2 （5.3%） 42 (33.3%）

<5 years
n. (%) 46 (51.7%) 15 (16.9%) 28 (31.5%) 89 (100%)
Male/Female 1.56 2 1.15 1.47
Culture positive (%) 25 (54.3%) 0 (0) 0 (0) 25 (28.1%)

Ultra-RTS positive (%) 28 (60.9%) 1 (6.7%） 0 (0) 29 (32.6%)

Ultra-Stool positive （%） 24 (52.2%) 0 (0) 1（3.6%） 25 (28.1%)

5-14 years
n. (%) 21 (56.8%) 6 (16.2%) 10 (27.0%) 37 (100%)
Male/Female 0.75 1 1.5 0.95
Culture positive (%) 15 (71.4%) 0 (0) 0 (0) 15 (40.5%)
Ultra-RTS positive (%) 15 (71.4%) 2 (33.3%) 0 (0) 17 (45.9%)

Ultra-Stool positive （%） 16 (76.2%) 0 (0) 1 (10.0%) 17 (45.9%)

RTS, respiratory tract sample; TB, tuberculos. * One cases with congenital heart
disease; one with bacterial pneumonia; one with inflammatory bowel disease;
one with leukemia. ** One with congenital heart disease; one with congenital
laryngeal chondrodysplasia.

20
Table 2. Diagnostic accuracy of assays for detection of paediatric
pulmonary tuberculosis against CRS

Result
Not
(positive TB Sensitivity 95% Specificity 95% 95% NPV 95% AUC
TB PPV (%)
& (n.) (%) CI (%) CI CI (%) CI *
(n.)
negative)

+ 46 0 0.41 0.91 0.92 0.36

Ultra-RTS 52.3% to 100.0% to 100.0% to 47.5% to 0.761

- 42 38
0.63 1.00 1.00 0.59

+ 40 2 0.35 0.82 0.84 0.32

Ultra-Stool 45.5% to 94.7% to 95.2% to 42.9% to 0.701

- 48 36
0.56 0.99 0.99 0.54

+ 32 0 0.26 0.91 0.91 0.33

Xpert-RTS 36.4% to 100.0% to 100.0% to 44.2% to 0.682

- 56 38
0.47 1.00 1.00 0.55

+ 40 0 0.35 0.91 0.91 0.33

Culture-RTS 45.5% to 100.0% to 100.0% to 44.2% to 0.727

- 48 38
0.56 1.00 1.00 0.55

+ 24 0 0.18 0.91 0.91 0.28

AFB-RTS 27.3% to 100.0% to 100.0% to 37.3% to 0.636

- 64 38
0.38 1.00 1.00 0.47

CRS, composite reference standard; RTS, respiratory tract sample; TB,

tuberculosis; CI, confidence interval; PPV, positive predictive value; NPV, negative
predictive value; AUC, area under the curve; AFB, acid-fast bacilli.

21
Table 3. Diagnostic accuracy of assays for detection of paediatric
pulmonary tuberculosis against Mtb culture

Result
Not
(positive TB Sensitivity 95% Specificity 95% PPV 95% NPV 95%
TB AUC *
& (n.) (%) CI (%) CI (%) CI (%) CI
(n.)
negative)

+ 31 15 0.62 0.73 0.52 0.80

Ultra-RTS 77.5% to 82.6% to 67.4% to 88.8% to 0.800

- 9 71
0.89 0.90 0.80 0.95

+ 28 14 0.53 0.74 0.50 0.76

Ultra-Stool 70.0% to 83.7% to 66.7% to 85.7% to

- 12 72 0.769
0.83 0.91 0.80 0.92

+ 26 6 0.48 0.85 0.64 0.76

Xpert-RTS 65.0% to 93.0% to 81.3% to 85.1% to 0.790

- 14 80
0.79 0.97 0.93 0.92

+ 20 4 0.34 0.89 0.63 0.71

AFB-RTS 50.0% to 95.4% to 83.3% to 80.4% to 0.727

- 20 82
0.66 0.99 0.95 0.88

Mtb, Mycobacterium tuberculosis; RTS, respiratory tract sample; TB, tuberculosis;

CI, confidence interval; PPV, positive predictive value; NPV, negative predictive
value; AUC, area under the curve; AFB, acid-fast bacilli.

22