# A multicountry evaluation of the Xpert MTB/RIF assay for the diagnosis of intrathoracic tuberculosis in children using alternative specimens (nasopharyngeal aspirate and stool): A prospective cohort study conducted in Madagascar, Ivory Coast and Cameroon (TB KIDS project)

Rindra Vatosoa Randremanana, Mathurin Tejiokem, Niaina

Rakotosamimanana, Valérie Donkeng Donfack, Verlaine Bolyse Mbouchong,

Mirella Randrianarisoa, Aina Harimanana, Jean Voisin Taguebue, Suzie

Tetang Ndiang, Valère Itchy, et al.

HAL Id: pasteur-04987379

https://pasteur.hal.science/pasteur-04987379v1

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Rindra Vatosoa Randremanana, Mathurin Tejiokem, Niaina Rakotosamimanana, Valérie Donkeng Donfack, Verlaine Bolyse Mbouchong, et al.. A multicountry evaluation of the Xpert MTB/RIF assay for the diagnosis of intrathoracic tuberculosis in children using alternative specimens (nasopharyngeal aspirate and stool): A prospective cohort study conducted in Madagascar, Ivory Coast and Cameroon (TB KIDS project). International Journal of Infectious Diseases, 2025, 151 (3), pp.107366. 10.1016/j.ijid.2024.107366. pasteur-04987379
# A multicountry evaluation of the Xpert MTB/RIF assay for the diagnosis of intrathoracic tuberculosis in children using alternative specimens (nasopharyngeal aspirate and stool): A prospective cohort study conducted in Madagascar, Ivory Coast and Cameroon (TB KIDS project)

<figure><img src="image_3.png" /></figure>

Rindra Vatosoa Randremanana<sup>1,#,*</sup>, Mathurin Tejiokem<sup>2,#</sup>, Niaina Rakotosamimanana<sup>1</sup>, Valérie Donkeng Donfack<sup>2</sup>, Verlaine Bolyse Mbouchong<sup>2</sup>, Mirella Randrianarisoa<sup>1</sup>, Aina Harimanana<sup>1</sup>, Jean Voisin Taguebue<sup>3</sup>, Suzie Tetang Ndiang<sup>4</sup>, Valère Itchy<sup>5</sup>, Annick Robinson<sup>6</sup>, Lovaniaina Ravelomanana<sup>7</sup>, Mbola Rakotomahefa<sup>8</sup>, Dina Ranoharison<sup>9</sup>, Man-Koumba Soumahoro<sup>10</sup>, Brigitte Gicquel<sup>11</sup>, Raymond N'Guessan<sup>8,##</sup>, Sara Eyangoh<sup>2,##</sup>, Voahangy Rasolofo<sup>1,##</sup>, on behalf of the TB-KIDS Study group<sup>***</sup>

## ARTICLE INFO

**Article history:**
Received 20 August 2024
Revised 13 December 2024
Accepted 13 December 2024

**Keywords:**
Tuberculosis
Children
Diagnosis
Xpert MTB/RIF
Nasopharyngeal aspirate
Stools

## ABSTRACT

**Objectives:** Tuberculosis (TB) diagnosis in children remains challenging due to the paucibacillary nature of specimens and the difficulty in obtaining suitable samples. The use of alternative samples like nasopharyngeal aspirate (NPA) and stools, alongside Xpert MTB/RIF testing, offers promising improvements. This study aimed to assess the diagnostic performance of the Xpert MTB/RIF test on NPA and stool samples for detecting intrathoracic TB in children from Madagascar, Cameroon, and Ivory Coast.

**Methods:** Children under 15 years with suspected intrathoracic TB were enrolled in hospitals in these countries' capitals. Samples for analysis included standard specimens (gastric aspirate or sputum), NPA, stools, with additional HIV serology, tuberculin skin test tests, and chest X-rays. We used a composite reference standard to estimate the accuracy of the Xpert MTB/RIF test with alternative samples.

**Results:** Of 1146 children analyzed, the sensitivity of Xpert MTB/RIF was 58.3% for NPA and 45.5% for stool samples, with a high specificity of more than 95%. The diagnostic performance of Xpert MTB/RIF with alternative samples did not differ according to age group or HIV status.

**Article history:**
Received 20 August 2024
Revised 13 December 2024
Accepted 13 December 2024

**Keywords:**
Tuberculosis
Children
Diagnosis
Xpert MTB/RIF
Nasopharyngeal aspirate
Stools

<footnote n="1"><sup>1</sup> Institut Pasteur de Madagascar, Antananarivo, Madagascar</footnote>

<footnote n="2"><sup>2</sup> Centre Pasteur du Cameroun, Yaoundé, Cameroun</footnote>

<footnote n="3"><sup>3</sup> Centre Mère et Enfant de la Fondation Chantal Biya, Yaoundé, Cameroun</footnote>

<footnote n="4"><sup>4</sup> Centre Hospitalier d'Essos, Yaoundé, Cameroun</footnote>

<footnote n="5"><sup>5</sup> Service de Pédiatrie, Centre Hospitalier Universitaire de Cocody, Abidjan, Côte d'Ivoire</footnote>

<footnote n="6"><sup>6</sup> Centre Hospitalier Universitaire Mère Enfant Tsaralalàna, Antananarivo, Madagascar</footnote>

<footnote n="7"><sup>7</sup> Centre Hospitalier Universitaire Mère Enfant Ambohimiandra, Antananarivo, Madagascar</footnote>

<footnote n="8"><sup>8</sup> Service de pédiatrie, Centre Hospitalier Universitaire Joseph Raseta Befelatanana, Antananarivo, Madagascar</footnote>

<footnote n="9"><sup>9</sup> Service d'imagerie médicale, Centre Hospitalier Universitaire Andohatapenaka, Antananarivo, Madagascar</footnote>

<footnote n="10"><sup>10</sup> Institut Pasteur de Côte d'Ivoire, Abidjan, Côte d'Ivoire</footnote>

<footnote n="11"><sup>11</sup> Institut Pasteur, Paris, France</footnote>

<footnote>* Corresponding author: (R. V. Randremanana).</footnote>

<footnote>E-mail address: rrandrem@pasteur.mg (R.V. Randremanana).</footnote>

<footnote># These authors contributed equally.</footnote>

<footnote>## These authors contributed equally.</footnote>

<footnote>*** See Members of the E. coli Study Group in Appendix.</footnote>
**Conclusions:** The findings support the World Health Organization's recommendation for using Xpert MTB/RIF with alternative samples in childhood TB diagnosis, underscoring its utility across different settings and HIV statuses.

© 2024 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

## Introduction

Tuberculosis (TB) remains a major global public health problem, with 10.6 million new cases reported worldwide in 2022 according to the World Health Organization (WHO) [1]. Geographically, 23% of the global TB burden in 2022 occurred in Africa. Children and adolescents under 15 years old represent approximately 12% of the total global TB cases, or 1.3 million children diagnosed with TB annually. More than 200,000 children lose their lives to TB each year [1,2]. Children under 5 years of age have a markedly higher risk of developing severe TB disease, including disseminated TB, even in immunocompetent patients, owing to their less developed immune systems. This risk is further elevated in immunocompromised and/or HIV-infected young patients [3].

However, the diagnosis of TB disease in children presents numerous challenges. Symptoms are frequently nonspecific, and microbiological confirmation is achieved in less than 50% of pediatric cases, as bacillary loads are typically low in biological samples from children [4,5]. In low-resource countries, diagnosis relies heavily on microscopic examination of respiratory samples, which is rapid but insensitive in paucibacillary childhood disease. Obtaining quality respiratory specimens can also be difficult. As a result, microscopy is positive in only 10-15% of probable pediatric TB cases [5]. Mycobacterial culture, though more sensitive, requires equipped laboratories often unavailable or inaccessible in resource-limited settings. Thus, anti-TB treatment is frequently initiated based on clinical diagnosis and/or clinical scoring systems without bacteriological confirmation [6].

Alternative diagnostic methods are clearly needed. The Xpert MTB/RIF assay has shown utility in high-burden countries [7–13], providing results in under 2 hours without extensive laboratory infrastructure. However, sample collection methods can influence diagnostic performance, and less invasive approaches like nasopharyngeal aspiration (NPA) and stool analysis warrant further study as alternatives to gastric aspiration (GA) [11–17]. In line with recent WHO recommendations, we conducted a multicenter cohort study evaluating Xpert MTB/RIF on NPA and stool samples for pulmonary TB diagnosis in children across three low-resource African countries with high TB incidence but varying HIV prevalence [1,18]. Our objective was to assess the diagnostic performance of Xpert MTB/RIF across alternative samples.

## Methods

### Study outline

This was a diagnostic accuracy study based on a multicenter prospective cohort carried out in the capitals of three countries in sub-Saharan Africa: Antananarivo (Madagascar), Abidjan (Ivory Coast), and Yaoundé (Cameroon). Children and young adolescents were enrolled in pediatric centers: in Antananarivo, at the Joseph Raseta Befelatanana University Hospital Center, the Mother-Child University Hospital Center of Tsaralalàna and the Mother and Child University Hospital Center of Ambohimiandra; in Abidjan, at the University Teaching Hospital of Cocody, Abidjan and at the General Hospital of Abobo North; in Yaoundé, at the Essos Hospi-

tal Center and the Centre Mère et Enfant de la Fondation Chantal Biya. Cases of suspected pulmonary TB were included consecutively from March 2016 to December 2018.

### Inclusion and exclusion criteria

Children younger than 15 years with suspected pulmonary TB were included if they had at least one of the following signs [19]: cough more than 2 weeks, weight loss or failure to thrive, lethargy or reduced playfulness, night sweats, dyspnea, symptoms suggesting extrapulmonary TB, household TB contact.

Children who had previously been treated for TB or had been on anti-TB treatment for more than a week were not included in the study.

### Data collected and clinical examinations upon inclusion

During the inclusion visit, data on medical history, recent exposure to TB, treatments, and history of disease were collected. Each participant had a complete clinical examination including nutritional status, cough characteristics, temperature, and symptoms suggestive of intra- and extrapulmonary TB, a tuberculin skin test (TST), and a frontal chest radiograph (Supplementary material S1).

### Sample collection and testing

For respiratory samples, we collected (i) two sputum samples in the morning upon waking up over 2 consecutive days if the child was aged 10 and over, (ii) two gastric aspirates (5 ml) in the morning on 2 consecutive days if the child was under 10 years old, in the fasted child since midnight the day before and (iii) one NPA performed by mechanical aspiration using a graduated suction tube inserted into the child's nostril.

One stool specimen was collected in a sterile plastic container. Stool samples were diluted with sucrose solution and vortexed. Mycobacterial identification was based on colony appearance, conventional biochemical tests, and immunochromatographic assay using SD bioline TB Ag MPT64, details are available in the Supplementary materials (S1). Stool specimens and NPA were collected from all age children.

The samples were analyzed at the Mycobacteria Laboratory of the three Pasteur Institutes in the three cities for Acid Fast Bacilli (AFB) microscopy and culture during 8 weeks on solid Lowenstein-Jensen medium and Coletsos medium and/or on Mycobacterial Growth Indicator Tube (MGIT) medium according to the standard procedures of each laboratory. Before mycobacterial culture, respiratory samples were decontaminated with sodium dodecyl sulfate. Microscopic examination was carried out to search for AFB in fresh samples after staining with auramine.

The Xpert MTB/RIF test was performed according to the manufacturer's instructions, by a technician who was blind to the microscopy and culture results.

A blood sample was collected from each child after pretest counseling with parents for HIV serology. If the test was positive and the child aged ≥18 months, cluster of differentiation 4 counts and HIV viral load were determined before combination antiretroviral therapy initiation according to the national current recom-
mendations. For children aged < 18 months with positive serolog-
ical tests, HIV DNA polymerase chain reaction was conducted for
confirmation.

Anti-TB treatment was initiated according to the doctor's diag-
nosis independently of the results of the microbiological analyses.

Data collected during follow-up visits

After inclusion, three visits were planned for children receiving anti-TB therapy: at month 1 (M₁), a clinical and radiological examination was carried out. At month 2 (M₂) and month 6 (M₆), clinical examination was done; a collection of sputum or GA for microscopy, culture, and Xpert MTB/RIF was performed if the child had a positive bacteriological examination at inclusion. Children not treated for TB had follow-up visits 2 months after inclusion during which signs/symptoms were recorded.

Sample size

The estimated sample size was 140 confirmed TB subjects and as many subjects with no TB, calculated based on assumptions on sensitivity and specificity at 90% and an accuracy of 5%.

Data management and statistical analysis

Data were collected using a standardized form, then entered in
duplicate and stored in a database in OpenClinica format (Open-
Clinica LLC, Waltham, MA, USA). A checking procedure was put
in place for outliers, inconsistencies, and missing data. Thereafter,
cleanings were launched periodically.

The distributions of the demographic, clinical, and paraclinical characteristics of the population were expressed as proportions for categorical variables and as a median with interquartile intervals for the quantitative variables.

The Xpert MTB/RIF test performance by using the alternative samples: NPA, stool, NPA stool was estimated with the sensitivity, specificity, positive/negative predictive values with 95% confidence intervals (CIs) calculated with the exact method. A composite reference standard based on symptoms, paraclinical characteristics, and microbiological results were used [20], thus included children were classified into one of the following categories: confirmed cases, unconfirmed cases, and unlikely cases [20] (Supplementary materials S2). For sensitivity estimation, both confirmed and unconfirmed cases were used as reference standards and we calculated the specificity among the unlikely TB cases.

We excluded Xpert NPA as part of the reference standard when it was evaluated as the index test to avoid incorporation bias.

We carried out subanalyses of the performance of the Xpert MTB/RIF test according to age and the HIV status of the included children.

Missing data on the index test/diagnostic samples and reference standard were excluded from the analysis, we did not perform imputation analysis.

Ethical considerations

The study protocol was approved by the Biomedical Research Ethics Committee of Madagascar, the National Ethics Committee for Human Health Research of Cameroon, the National Ethics and Research Committee of Côte d'Ivoire, and the Clinical Research Committee of the Institut Pasteur in Paris. The study was performed in accordance with the 2013 Declaration of Helsinki and Good Clinical Practice guidelines.

Written informed consent was obtained by at least one parent
or legal guardian of the child, and the assent of children older than
7 years was also collected.

Results

Study population, clinical, and paraclinical characteristics

A total of 1165 children clinically suspected of intrathoracic TB (500 in Madagascar, 394 in Ivory Coast, 271 in Cameroon) were enrolled; 1146 (98.4%), for whom at least one GA or one sputum sample was obtained. Among them, 616 (53.8%) were male.

The characteristics of children included with at least one stan-
dard sample (n = 1146) are described in Table 1. The median age
was 3.7 years (interquartile range [1.3-8.7]). The proportion of mal-
nourished children was 47.3% (n = 487), and 11.6% (n = 133) were
HIV-positive. More than a quarter (26.2%, n = 300) of children had
known exposure to TB; 69% (n = 198) of them had contact with
TB in the 12 months before inclusion in the study.

The clinical and paraclinical characteristics of children are shown in Table 2. The three most frequently observed symptoms were cough (84.2%, 965/1146), fever (83.1%, 951/1146), and weight loss (73.9%, 847/1,146). Results of TST were available in 82.7% of the included children, and 24.6% (n = 225) had a positive test. During this study, 64.3% (n = 737) of the children had chest X-ray results, among them 31.7% (n = 234) had radiological images in favor of intrathoracic TB. Anti-TB treatment was initiated in 31.6% of children (n = 362).

Bacteriological results

Of the 1146 children with standard sample (i.e., sputum or GA), 1102 (96.1%) had an NPA sample, 1058 (92.3%) had a stool sample (Figure 1) and 1136 children had one NPA sample or one stool specimen. Around 90% (n = 1035) of children were able to provide more than one standard sample, 12.9% (148/1146) provided more than one sputum sample, and 77.4% (887/1146) had more than one GA. Among children with at least one standard sample, 13.2% (151/1146) had at least one positive bacteriological test (microscopy or culture or Xpert MTB/RIF). Figure 1 and Supplementary Materials S3 show details of bacteriological results for different biological samples.

Follow-up of children

Among the 1146 children, 75 (6.4%) died during their hospital-
ization (29 treated with anti-TB and 46 not treated with anti-TB).
Among the 333 children treated with anti-TB drugs and alive when
they left the hospital, 268 (81.5%) had a visit in the 1st month.
The proportion of children who performed the 2nd and 6th month
follow-up visits was respectively 79.7% and 72.7%. Among those
who did not receive anti-TB therapy, 76.3% (n = 563) carried out
their follow-up in the 2nd month. In total, the proportion of deaths
was 11.8%. (n = 135) and 5.6% of deaths (n = 60) occurred during
the follow-up (Supplementary materials S4).

Tuberculosis status of children according to the definitions of Graham
et al. [20]

Among those who had at least one standard specimen (sputum or GA), the proportion of confirmed TB cases was 13.7% (157/1146), those of unconfirmed TB and unlikely TB, were 18.0% (206/1146), and 41.5% (476/1146) respectively. TB status of 26.8% of children (307/1146) could not be determined. The main reasons were chest X-ray not done (149/307), TST not performed or not read (110/307), and lack of data on previous exposure to TB (23/307). There were 15 deaths among the unclassified cases. TB status of children according to the index samples (NPA, stools, NPA or stools) Xpert MTB/RIF assay are shown in Table 3.
<table><caption>**Table 1**

General characteristics of children included in the analysis (N = 1146).</caption><thead><tr><th>Characteristics</th><th>n (%)</th></tr></thead><tbody><tr><td><strong>Sex</strong></td><td></td></tr><tr><td>Male</td><td>616 (53.8%)</td></tr><tr><td>Female</td><td>530 (46.2%)</td></tr><tr><td><strong>Age in years (median [1st-3rd quartile])</strong></td><td>3.7 (1.3-8.7)</td></tr><tr><td><strong>Age &lt; 5 years</strong></td><td></td></tr><tr><td>Yes</td><td>695 (60.6%)</td></tr><tr><td>No</td><td>451 (39.4%)</td></tr><tr><td><strong>Nutritional status<sup>a</sup></strong></td><td></td></tr><tr><td>Severely malnourished</td><td>285 (27.7%)</td></tr><tr><td>Moderately malnourished</td><td>202 (19.6%)</td></tr><tr><td>Non-malnourished</td><td>542 (52.7%)</td></tr><tr><td>Not specified</td><td>117</td></tr><tr><td><strong>HIV status</strong></td><td></td></tr><tr><td>HIV-positive</td><td>133 (12.0%)</td></tr><tr><td>HIV-negative</td><td>976 (88.0%)</td></tr><tr><td>Test not done</td><td>37</td></tr><tr><td><strong>Proof of Mycobacterium bovis bacillus Calmette-Guérin vaccination (by booklet or scar)</strong></td><td></td></tr><tr><td>Yes</td><td>860 (75.8%)</td></tr><tr><td>No</td><td>275 (24.2%)</td></tr><tr><td>Missing data</td><td>11</td></tr><tr><td><strong>Exposure to TB</strong></td><td></td></tr><tr><td>Yes</td><td>300 (26.2%)</td></tr><tr><td>No</td><td>767 (66.9%)</td></tr><tr><td>Not known</td><td>79 (6.9%)</td></tr><tr><td><strong>Exposure to TB less than 12 months before the survey<sup>b</sup></strong></td><td></td></tr><tr><td>Yes</td><td>198 (66.0%)</td></tr><tr><td>No</td><td>89 (29.7%)</td></tr><tr><td>Not known</td><td>13 (4.3%)</td></tr></tbody></table>

BMI, body mass index

<sup>a</sup> Severely malnourished: infants 5-14 years with BMI for age <-3SD or infants ≤5 years with z-score weight for length <-3SD or with z-score height for age<-3SD; moderately malnourished: infants 5-14 years with BMI for age between -2SD and -3SD or infants ≤5 years with z-score weight for length between -2SD and -3SD or with z-score height for age between -2SD and -3SD; non-malnourished: infants 5-14 years with BMI for age >-2SD, infants ≤5 years with z-score weight for length >-2SD or with z-score height for age >-2 SD

<sup>b</sup> Exposure to TB: history of TB in the surroundings.

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Flow chart of children according to the standard and alternative samples available (diagram by country in Supplementary Materials). GA, gastric aspiration; NPA, nasopharyngeal aspiration.</figcaption></figure>
<table><caption>**Table 2**
Clinical and paraclinical characteristics of children (N = 1146).</caption><thead><tr><th>Characteristics</th><th>No. (%)</th></tr></thead><tbody><tr><td><strong>Symptoms</strong></td><td></td></tr><tr><td>Cough</td><td></td></tr><tr><td>Yes</td><td>965 (84.2%)</td></tr><tr><td>No</td><td>181 (15.8%)</td></tr><tr><td>Cough > 14 days<sup>a</sup></td><td></td></tr><tr><td>Yes</td><td>547 (57.3%)</td></tr><tr><td>No</td><td>407 (42.7%)</td></tr><tr><td>Not known</td><td>11</td></tr><tr><td>Fever (>38°C)</td><td></td></tr><tr><td>Yes</td><td>951 (83.1%)</td></tr><tr><td>No</td><td>194 (16.9%)</td></tr><tr><td>Not available</td><td>1</td></tr><tr><td>Fever > 7 days<sup>b</sup></td><td></td></tr><tr><td>Yes</td><td>729 (77.7%)</td></tr><tr><td>No</td><td>209 (22.3%)</td></tr><tr><td>Not known</td><td>13</td></tr><tr><td><strong>Weight loss</strong></td><td></td></tr><tr><td>Yes</td><td>847 (73.9%)</td></tr><tr><td>No</td><td>299 (26.1%)</td></tr><tr><td><strong>Paraclinical data</strong></td><td></td></tr><tr><td>Tuberculin test performed</td><td></td></tr><tr><td>Yes</td><td>912 (82.7%)</td></tr><tr><td>No</td><td>191 (17.3%)</td></tr><tr><td>Missing data</td><td>43</td></tr><tr><td>Positive tuberculin test<sup>c</sup></td><td></td></tr><tr><td>Yes</td><td>225 (25.8%)</td></tr><tr><td>No</td><td>647 (74.2%)</td></tr><tr><td>Missing data</td><td>40</td></tr><tr><td>Children with chest X-ray suggestive of tuberculosis<sup>d</sup></td><td></td></tr><tr><td>Yes</td><td>234 (31.7%)</td></tr><tr><td>No</td><td>501 (68.0%)</td></tr><tr><td>Not known</td><td>2 (0.3%)</td></tr></tbody></table>

Performance of the Xpert MTB/RIF assay using nasopharyngeal aspirate and stool specimens

Table 3 shows the diagnostic characteristics of the Xpert MTB/RIF test with three types of specimens (NPA, stool, and NPA or stool), taking both the confirmed TB and the unconfirmed TB groups as a positive reference and the unlikely TB group for the estimation of the specificity. The sensitivity of Xpert MTB/RIF was 58.3% (95% CI: 49.4-66.8) with NPA specimens, 45.5% (95% CI: 36.9-54.3) with stool specimens, and 63.2% (95% CI: 54.8-71.1) when considering the results of one of the two samples (NPA or stool). The sensitivity of Xpert was estimated against microbiological confirmation as the reference standard.

The specificity was 99.8% (95% CI: 98.7-100) and 96.5% (95% CI: 94.2-98.1) for the Xpert MTB/RIF test with NPA and stools, respectively.

and 96.6% (95% CI: 94.5-98.1) for the Xpert MTB/RIF test using NPA or stools.

The positive predictive value (PPV) of the NPA Xpert MTB/RIF assay was 95% (95% CI: 88-99%), and the negative predictive value (NPV) was 94% (95% CI: 92-95%). With stool samples, the PPV of the Xpert MTB/RIF test was 63% (95% CI: 53-73%), and the NPV was 91% (95% CI: 89-93%).

The PPV and NPV of the Xpert MTB/RIF test using NPA or stool were 69% (95% CI: 60-77) and 94% (95% CI: 93-97), respectively.

Xpert MTB/RIF test performance using alternative specimens according to the age of the child

Table 4 shows the performance of the Xpert MTB/RIF test using the alternative samples according to patient age. The results showed that regardless of the type of biological samples, the performance of the test did not differ significantly according to the age groups considered.

Xpert MTB/RIF test performance using alternate specimens according to HIV status

No significant difference was observed in the performance of the Xpert MTB/RIF test using the alternative samples between the HIV-positive and HIV-negative groups. The sensitivity of the Xpert MTB/RIF test was 53.8% (95% CI: 25.1-80.8) for NPA in HIV-positive subjects and 60% (95% CI: 50.4-69.0) in HIV-negative subjects. Using stool samples, the sensitivity of the Xpert MTB/RIF test was 58.3% (95% CI: 27.7-84.8) in HIV-positive subjects and 44.1% (34.9-53.5) in HIV-negative subjects. Using NPA or stool specimens, the sensitivity of the Xpert MTB/RIF test was 61.5% (95% CI: 31.6-86.1) in HIV-positive subjects and 64.6% (55.6-72.8) and HIV-negative subjects (Table 5).

The specificities of the Xpert MTB/RIF test with NPA, stool, NPA, and/or stools were all 100% in subjects who had a positive HIV test. In those who had a negative HIV test, the specificities of the Xpert MTB/RIF test were 99.7% (95% CI: 98.5-100), 96.3% (95% CI: 93.8,-98) and 96.4% (95% CI: 94.1-98) with NPA, stools and NPA or stool, respectively.

Discussion

This study aimed to assess the performance of the Xpert MTB/RIF on less invasive samples such as NPA and stool for pulmonary TB diagnosis in children in three low-resource countries. We reported promising results and found that alternative samples could be used to enhance the diagnosis of TB in children even if its sensitivities were limited.

<table><caption>**Table 3**
Diagnostic performance of alternative samples Xpert MTB/RIF assay compared to a composite reference standard [20].</caption><thead><tr><th rowspan="2">Xpert MTB/RIF</th><th colspan="2">Sensitivity</th><th>Specificity</th></tr><tr><th>Confirmed TB<sup>a</sup><br>n/N; % (95% CI)</th><th>Unconfirmed TB<sup>a</sup><br>n/N; % (95% CI)</th><th>Unlikely TB<sup>a</sup><br>n/N; % (95% CI)</th></tr></thead><tbody><tr><td>NPA<sup>b</sup></td><td>77/132; 58.3% (49.4-56.8)</td><td>2/191; 1.0% (0.1-3.7)</td><td>424/425; 99.8% (98.7-100)</td></tr><tr><td>Stools<sup>c</sup></td><td>61/134; 45.5% (36.9-54.3)</td><td>11/176; 6.2% (3.2-10.9)</td><td>387/401; 96.5% (94.5-98.1)</td></tr><tr><td>NPA or stools<sup>d</sup></td><td>91/144; 63.2% (54.8-71.1)</td><td>15/199; 7.5% (4.3-12.1)</td><td>426/441; 96.6% (94.5-98.1)</td></tr></tbody></table>

CI, confidence interval; NPA, nasopharyngeal aspirate; TB, tuberculosis.

a For the evaluation of the sensitivity of the Xpert test with NPA, the reference was confirmed TB cases with sputum or GA (Xpert test or culture positive with sputum or GA) and unconfirmed TB [20]. For specificity, we estimated it among unlikely TB [20]. For the evaluation of the sensitivity of the Xpert test with stool specimens, the reference was confirmed TB cases with sputum or GA or NPA (Xpert test or culture positive with sputum or GA or NPA) and unconfirmed TB cases [20]. For specificity, the reference was composed of unlikely TB cases [20].

b For the estimation of accuracy of NPA, 1010 children was included in the analysis, eight did not have NPA samples.

c We calculated the diagnostic performance of stools with 1066 children, nine children were not able to provide stool samples.

d We estimated the accuracy of NPA or stools with 939 children.

<footnote>a n = 965.</footnote>

<footnote>b n = 951.</footnote>

<footnote>c n = 912.</footnote>

<footnote>d n = 737.</footnote>
<table><caption>**Table 4**
Performance of Xpert MTB/RIF by age group and specimen type in children with suspected intrathoracic TB.</caption><thead><tr><th rowspan="2">Xpert MTB/RIF</th><th colspan="2">Sensitivity</th><th rowspan="2">Specificity<br/>Unlikely TB<sup>a</sup><br/>n/N; % (95% CI)</th></tr><tr><th>Confirmed TB<sup>a</sup><br/>n/N; % (95% CI)</th><th>Unconfirmed TB<sup>a</sup><br/>n/N; % (95% CI)</th></tr></thead><tbody><tr><td colspan="4"><strong>&lt;5 years</strong></td></tr><tr><td>NPA</td><td>42/71; 59.2% (46.8-70.7)</td><td>1/95; 1.1% (0.0-5.7)</td><td>275/276; 99.6% (98.0-100)</td></tr><tr><td>Stools</td><td>35/75; 46.7% (35.1-58.6)</td><td>7/85; 8.2% (3.4-16.2)</td><td>248/259; 95.8% (92.5-97.9)</td></tr><tr><td>NPA or stools</td><td>49/75; 65.3% (53.5-76.0)</td><td>8/99; 8.1% (3.6-15.3)</td><td>275/287; 95.8% (92.8-97.8)</td></tr><tr><td colspan="4"><strong>≥5 years</strong></td></tr><tr><td>NPA</td><td>35/61; 57.4% (44.1-70.0)</td><td>1/96; 1.0% (0.0-5.7)</td><td>149/149; 100% (97.6-100)</td></tr><tr><td>Stools</td><td>26/59; 44.1% (31.2-57.6)</td><td>4/91; 4.4% (1.2-10.9)</td><td>139/142; 97.9% (94.0-99.6)</td></tr><tr><td>NPA or stools</td><td>42/69; 60.9% (48.4-72.4)</td><td>7/100; 7.0% (2.9-13.9)</td><td>151/154; 98.1% (94.4-99.6)</td></tr></tbody></table>

CI, confidence interval; NPA, nasopharyngeal aspirate; TB, tuberculosis.

<sup>a</sup> For the evaluation of the sensitivity of the Xpert test with NPA, the reference was composed of confirmed TB cases with sputum or GA (Xpert test or culture positive with sputum or GA) and unconfirmed TB [20]. For specificity, we estimated it among unlikely TB [20]. For the evaluation of the sensitivity of the Xpert test with stool specimens, the reference was composed of unconfirmed TB cases with sputum or GA or NPA (Xpert test or culture positive with sputum or GA or NPA) and unconfirmed TB cases [20]. For specificity, the reference was composed of unlikely TB cases [20].

<table><caption>**Table 5**
Performance of Xpert MTB/RIF by HIV status and alternative specimen type in children with suspected intrathoracic TB.</caption><thead><tr><th rowspan="2">Xpert MTB/RIF</th><th colspan="2">Sensitivity</th><th rowspan="2">Specificity<br/>Unlikely TB<sup>a</sup><br/>n/N; % (95% CI)</th></tr><tr><th>Confirmed TB<sup>a</sup><br/>n/N; % (95% CI)</th><th>Unconfirmed TB<sup>a</sup><br/>n/N; % (95% CI)</th></tr></thead><tbody><tr><td colspan="4"><strong>Positive HIV</strong></td></tr><tr><td>NPA</td><td>7/13 ; 53.8% (25.1-80.8)</td><td>0/53 ; 0.0% (0.0-6.7)</td><td>34/34 ; 100% (89.7-100)</td></tr><tr><td>Stools</td><td>7/12 ; 58.3% (27.7-84.8)</td><td>3/48 ; 6.2% (1.3-17.2)</td><td>34/34 ; 100% (89.7-100)</td></tr><tr><td>NPA or stools</td><td>8/13 ; 61.5% (31.6-86.1)</td><td>3/53 ; 5.7% (1.2-15.7)</td><td>35/35 ; 100% (90.0-100)</td></tr><tr><td colspan="4"><strong>Negative HIV</strong></td></tr><tr><td>NPA</td><td>69/115 ; 60% (50.4-69.0)</td><td>1/136 ; 0.7% (0.0-4.0)</td><td>378/379 ; 99.7% (98.5-100)</td></tr><tr><td>Stools</td><td>52/118 ; 44.1% (34.9-53.5)</td><td>8/127 ; 6.3% (2.8-12.0)</td><td>343/356 ; 96.3% (93.8-98.0)</td></tr><tr><td>NPA or stools</td><td>82/127 ; 64.6% (55.6-72.8)</td><td>11/144 ; 7.6% (3.9-13.3)</td><td>380/394 ; 96.4% (94.1-98.0)</td></tr></tbody></table>

CI, confidence interval; NPA, nasopharyngeal aspirate; TB, tuberculosis.

<sup>a</sup> For the evaluation of the sensitivity of the Xpert test with NPA, the reference was composed of confirmed TB cases with sputum or GA (Xpert test or culture positive with sputum or GA) and unconfirmed TB [20]. For specificity, we estimated it among unlikely TB [20]. For the evaluation of the sensitivity of the Xpert test with stool specimens, the reference consisted of confirmed TB cases with sputum or GA or NPA (Xpert test or culture positive with sputum or GA or NPA) and unconfirmed TB cases [20]. For specificity, the reference was composed of unlikely TB cases [20].

The specificities exceeded 90% and sensitivities even if modest, were comparable to those of culture tests conducted on standard samples [21]. Our composite reference standard included many tests and diagnostic samples that have the chance to bacteriologically confirm, this may reduce the sensitivities of the index test in diagnostic samples.

The study's findings on the sensitivity of the Xpert MTB/RIF test varied by sample type. NPA samples showed a sensitivity of 58.3%, which aligns with results from a systematic review which found a sensitivity of 47.5% (27.6-65.1%) [22], previous studies conducted in Kenya with a sensitivity of 66% (55-77%) [23] and in South Africa, though the latter reported a significantly lower sensitivity of 30% (16.7-47.9%) [8]. Stool samples, on the other hand, demonstrated a lower sensitivity of 45.5%. Literature reports highlighted the heterogeneity of sensitivity of the stool Xpert test [24-26] which might be related to several factors, such as sample volume, treatment process or HIV status. However, a recent systematic review and meta-analysis on the performance of stool-based molecular tests on pediatric TB diagnosis reported no differences in stool Xpert accuracy when using a simplified or more complex processing method [27]. Using a simplified stool processing method is encouraged mainly in resource-challenged settings.

Exploring alternative methods, such as rectal swabs, which have been reported for diagnosing other diseases like SARS-CoV-2 [28,29], could offer practical benefits. Besides employing two biological samples (NPA and stool) instead of one improved the diagnostic sensitivity of the Xpert MTB/RIF test to 63.2%, demonstrating that multiple sample types can enhance diagnostic accuracy.

The study revealed that the Xpert MTB/RIF test's sensitivity was significantly higher than that of microscopy, the most commonly used diagnostic method in developing countries [21]. Despite the promising sensitivity rates of the test when using NPA or stool samples, comparable to culture results, the performance of alternative samples Xpert MTB/RIF assay was poor among unconfirmed TB, indicating that this category of children which had negative microbiological results might have TB paucibacillary disease.

Diagnostic yield is an additional metric that can be included when assessing TB tests with limited sensitivity [30] such as with alternative samples Xpert MTB/RIF assay. In our study, the diagnostic yield of stool Xpert is comparable to those of Xpert in standard samples which were at 10.4% (Supplementary materials S3). Stool is an easily accessible specimen, our study suggested that it represented an increase of 8% in microbiological confirmation. Diagnostic yield is a more pragmatic indicator as it considers test accuracy and specimen availability. It could be used in combination with accuracy study indicators for the evaluation of TB tests or diagnostic samples.

The study's strengths lie in its large participant number, making it one of the first prospective studies of this scale in Africa to examine the performance of the Xpert MTB/RIF test on alternative samples. The inclusion of countries with different HIV contexts provided valuable insights into the test's diagnostic performance across varied settings. Notably, the sensitivity of the Xpert MTB/RIF test on NPA samples did not differ significantly according to HIV status, although stool samples exhibited higher sensitivity in HIV-seropositive children.
However, the study faced several limitations, including challenges in ensuring participant follow-up, particularly in Madagascar and Ivory Coast. Additionally, nearly a quarter of the children's TB status could not be definitively determined, reflecting the practical difficulties in conducting paraclinical examinations and the unavailability of results. This issue mirrors the real-world challenges faced in developing countries, where logistical barriers and other priorities can hinder access to healthcare services. Despite these obstacles, the study achieved its objectives by demonstrating the potential of less invasive sample collection methods to improve the diagnosis of intrathoracic TB in children. Secondly, we analyzed data for all countries as though they were one center to improve study's external validity, thus inter-center variability might exist. We implemented standard operating procedures for microbiological sample management and data collection to minimize potential inter-center variability. We found that performance indicators did not differ between the three countries. Thirdly, the composite reference we used is specifically related to intrathoracic TB, however, we applied it to a cohort of children that may have extrapulmonary symptoms. Lastly, in recent years, Xpert MTB/RIF Ultra was developed to enhance molecular diagnostic of TB, it seems to have a slightly better sensitivity than Xpert but it was not yet available in our study countries when we implemented the current study.

## Conclusion

This study highlights the utility of the Xpert MTB/RIF test with alternative samples like NPA and stool in enhancing the bacteriological diagnosis of TB in children. The findings advocate for the adoption of less invasive sample collection methods, which could significantly benefit TB diagnosis in pediatric populations, especially in resource-limited settings. Given the test's comparable diagnostic performance to culture and its superiority over microscopy, the Xpert MTB/RIF test represents a valuable diagnostic tool in the fight against TB. Further research is warranted to explore the feasibility and diagnostic accuracy of NPA in low-resource environments, potentially revolutionizing TB diagnosis in children worldwide.

This aligns with WHO's 2022 guidelines, recommending Xpert MTB/RIF as the primary test for pediatric pulmonary TB and rifampicin resistance, over conventional smear microscopy, culture, and phenotypic DST [2].

## Declarations of competing interest

The authors have no competing interests to declare.

## Funding

This work was supported by the Fondation TOTAL [Convention de mécénat entre La Fondation d'entreprise Total, l'Institut Pasteur, l'Institut Pasteur de Madagascar, l'Institut Pasteur de Côte d'Ivoire et le Centre Pasteur du Cameroun, du 21/07/2014].

## Ethical approval

The study protocol was validated by the Biomedical Research Ethics Committee of Madagascar (015-MSNPA/CE of 26/03/2014), the National Ethics Committee for Human Health Research of Cameroon (2015/07/619/CE/CNERSH/SP), the National Ethics and Research Committee of Côte d'Ivoire (44/MSLS/CNER-dkn), and the Clinical Research Committee of the Institut Pasteur in Paris (2013-25 of 05/11/2015).

## Acknowledgments

The authors acknowledge the hospital staff of the pediatric departments from the Joseph Raseta Befelatanana University Hospital Center, the Mother-Child University Hospital Center of Tsaralàna, and the Mother and Child University Hospital Center of Ambohimandra in Antananarivo; the University Teaching Hospital of Cocody and at the General Hospital of Abobo North in Abidjan; the Essos Hospital Center and the Centre Mère et Enfant de la Fondation Chantal Biya in Yaoundé. The authors would like to thank the laboratory staff of the Unité des Mycobactéries at Institut Pasteur de Madagascar, the Service de Mycobactériologie at the Centre Pasteur du Cameroun, and the Unité des Mycobactéries Tuberculeuses et Atypique at the Institut Pasteur de Côte d'Ivoire for their participation.

## Author contributions

RVR : Study design, supervision of data collection, Data analysis, Writing and final approval; MT : Study design, Data collection, data interpretation, final approval; NR : supervising lab analysis, final approval; VDD : supervising lab analysis, final approval; VBM : Data collection, final approval; MR : Data analysis, Writing, final approval; AH : Data collection, final approval; JVT : Data collection, final approval; SNT : Data collection, final approval; VI : Data collection, final approval; AR ,LR ,MR : Data collection, final approval; DR : Data interpretation, final approval; MKS : Data collection, final approval; BG : study conception, final approval; RN ,SE ,VR : study conception, supervision of data collection, writing and final approval.

## Appendix. TB-KIDS study group

The authors+ Emery Flavien Akpafi¹⁰, Marius Irie Bi Irie¹⁰, Anabelle Bai-Orsot¹⁰, Jaudel Francis Yuya Septoh², Serge Abogo⁴, Mamy Serge Raherison¹, Andrianantenaina Rakotoson¹, Vaomalala Raharimanga¹, Patrice Piola¹, Paulo Ranaivomanana¹, Jean-Marc Collard¹,¹¹, Gabrielle Prisca Emmylou Andrianah⁹, Turibio Razafindranaivo¹², Reziky Tiandraza Mangahasimbola¹, Kathleen Victoir¹¹

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ijid.2024.107366.

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