# Contribution and acceptability of bacteriological collection tools in the diagnosis of tuberculosis in children infected with HIV

Bintou Sanogo, Pegdewende Eliane Kiema, Makoura Barro, Sahoura Fatima Nacro, Saga Alain Ouermi, Philippe Msellati, Boubacar Nacro

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Bintou Sanogo, Pegdewende Eliane Kiema, Makoura Barro, Sahoura Fatima Nacro, Saga Alain Ouermi, et al.. Contribution and acceptability of bacteriological collection tools in the diagnosis of tuberculosis in children infected with HIV. Journal of Tropical Pediatrics, 2022, 67 (2), fmab027 [9 p.]. 10.1093/tropej/fmab027. hal-03774911

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# Contribution and Acceptability of Bacteriological Collection Tools in the Diagnosis of Tuberculosis in Children Infected with HIV

Bintou Sanogo, MD, MSc<fnref n="1" />,<fnref n="2" />, Pegdewende Eliane Kiema ID, MD<fnref n="3" />, Makoura Barro, MD<fnref n="1" />,<fnref n="2" />, Sahoura Fatimata Nacro, MD<fnref n="3" />, Saga Alain Ouermi, MD<fnref n="4" />, Philippe Msellati, PhD<fnref n="5" /> and Boubacar Nacro, MD<fnref n="2" />

Correspondence: Pegdewende Eliane Kiema, University Center Pediatric Charles de Gaulle, Ouagadougou, Burkina Faso. Tel: 76-92-2995.

Fax: 70-43-9559. E-mail: <kiemeliane.ek@gmail.com>.

## ABSTRACT

**Objective:** The objective of this study is to evaluate the feasibility and tolerability of new bacteriological samples to diagnose tuberculosis (TB) in HIV-infected children.

**Method and patients:** HIV1-infected children with suspicion of TB in University Hospital Sourô Sanon (Burkina Faso) were included in a prospective cohort study. Children underwent three gastric aspirates (GA) if aged <4 years; two GA, one string test (ST) if aged 4–9 years and three sputum, one ST if aged 10–13 years. All children underwent one nasopharyngeal aspirate (NPA) and one stool sample. To assess feasibility and tolerability of procedures, adverse events were identified and pain was rated on different scales. Samples were tested by microscopy, culture, GeneXpert® (Xpert®).

**Results:** Sixty-three patients were included. Mean age was 8.92 years, 52.38% were females. Ninety-five GA, 67 sputum, 62 NPA, 60 stool and 55 ST had been performed. During sampling, the main adverse events were cough at 68/95 GA and 48/62 NPA; sneeze at 50/95 GA and 38/62 NPA and vomiting at 4/55 ST. On the behavioral scale, the average pain score during collection was 6.38/10 for GA; 7.70/10 for NPA and 1.03/10 for ST. Of the 31 cases of TB, bacteriological confirmation was made in 12 patients.

**Conclusion:** ST, stool is well-tolerated alternatives specimens for diagnosing TB in children. NPA has a poor feasibility and tolerability in children.

**KEYWORDS:** tuberculosis, HIV, child, NPA, string test, stool

<footnote n="1">¹Higher Institute of Health Sciences (INSSA), Nazi Boni University (UNB), 01 BP 1091 Bobo-Dioulasso 01, Burkina Faso</footnote>

<footnote n="2">²Department of Pediatrics, University Hospital Center Souro Sanou (CHUSS), 01 BP 676 Bobo-Dioulasso, Burkina Faso</footnote>

<footnote n="3">³Universitary Center Pediatric Charles de Gaulle, 01 BP 1198 Ouagadougou 01, Burkina Faso</footnote>

<footnote n="4">⁴Pediatrics Department, Regional Teaching Hospital of Ouahigouya, Burkina Faso</footnote>

<footnote n="5">⁵Research Institute for Development, University of Montpellier 1, UMI 233 Montpellier, France</footnote>
# INTRODUCTION

In 2018, the World Health Organization (WHO) estimated the annual burden of tuberculosis (TB) in world at approximately 10 million cases, including 1.1 million children [1]. TB is the leading cause of death among HIV-infected children worldwide, accounting for one-third of all deaths in this group [2, 3]. Childhood TB remains under-diagnosed and underreported, even more in children infected with HIV [4, 5].

Microbiological confirmation of pulmonary tuberculosis (PTB) in children remains challenging. Collection of specimens for testing, such as induced sputum (IS) or gastric aspirate (GA), is perceived to be invasive and complex [5, 6]. GA is unpleasant, relatively invasive, often requires hospitalization; sputum sample is not feasible for young children [5, 7]. Consequently, the diagnosis of pediatric TB often relies on a combination of clinical judgment and radiological findings, prompting initiation of treatment without clear laboratory evidence [5, 8–12]. It is, therefore, necessary to find new tools for collecting bacteriological samples, as recommended by the WHO since 2007 [8]. One approach to improving TB diagnosis in children is to collect multiple specimens of different types with the assumption that the yield would be cumulative. Potentially useful specimen types include GA, IS, nasopharyngeal aspirates (NPA), stool, urine and the string test (ST) [4, 6, 13–15].

Several studies have evaluated the performance of these alternative bacteriological samples for the diagnosis of TB in children, but little data exist on their feasibility and tolerability.

In this study, we proposed to evaluate the feasibility and tolerability of alternative bacteriological collection methods, such as NPA, ST and stool sample in the diagnosis of TB in children infected with HIV.

# PATIENTS AND METHODS

We analyzed data of University Hospital Centre Souro Sanou (CHUSS) of Bobo-Dioulasso, participating in a multicentric prospective cohort study ANRS 12229 (Agence Nationale de Recherche sur le Sida): PAANTHER 01 (Pediatric Asian African Network for Tuberculosis and HIV Research). This multicentric study was simultaneously conducted at

seven other hospitals sites in Cambodia, Cameroon and Vietnam. From 5 December 2012 to 4 November 2014, all HIV1-infected children aged 0–13 years, and suspected to have pulmonary and extra-PTB, were included in the study based on at least one of the following signs: persistent cough for more than 2 weeks, persistent fever for at least 2 weeks, delayed weight clearly documented, failure of broad spectrum antibiotic treatment for pulmonary infection and suggestive anomaly of chest radiography. Children with a history of treatment for TB initiated in the last 2 years prior to the study were not included.

# Procedures

All nurses in the study have been trained in blood and bacteriological sampling procedures and in the evaluation of the feasibility and tolerability of the different sampling methods.

Bacteriological samples were taken using standard and alternative collection methods. In children under 4 years of age, three GA (on 3 days), for those aged 4–9 years 2 GA (on 2 days), one ST, among children over 10 years of age, three sputum sample (2 days) and one ST were collected. Among these, GA was performed in those unable to produce enough sputum. All children underwent one NPA and one stool sample.

NPA, ST and stool sample were the alternative collection methods. For collection of GA, sputum sample and ST, children were kept fasting overnight.

In addition, all children received a complete physical examination, chest X-ray, abdominal ultrasound, baseline blood sample including hemogram, HIV viral load and CD4 count and tuberculin skin test.

## Gastric aspirate

GA was performed using a nasogastric tube in the morning after overnight fasting. GAs were neutralized with 8% sodium bicarbonate.

## Nasopharyngeal aspirate

Nasopharyngeal aspiration was performed after GA, without prior nasal instillation, using a mucus aspirator connected to a suction device in order to stimulate the cough reflex and obtain 2–5 ml of mucus.
Procedure was performed under peripheral oxygen saturation monitoring with an oximeter.

## String test

The ST was performed using the pediatric Entero-Test (HDC Corporation, Milpitas, CA, USA). The gelatin capsule was swallowed, unraveling a 90-cm absorbent nylon string and the nonabsorbent part of the string was taped to the cheek with plaster.

After an intragastric downtime of 2 h, the string was retrieved and the absorbent part was deposited in a falcon tube containing 3 ml 0.9% saline [4].

## Feasibility and tolerability

To assess the feasibility of GA, ST and NPA, adverse events were recorded and pain was assessed using three different scales. The adverse events recorded by the nurses during the different procedures were

* for NPA and GA: coughing, sneezing, nausea, vomiting, trauma and nose bleeding, bradycardia (cardiac frequency <60/mn), dyspnea, oxygen desaturation (<90%) and

* for the ST: the occurrence of nausea or vomiting.

Tolerability, pain and discomfort during NPA, GA and ST had been rated before, during and after the procedures, by using different tools usually used to assess the perception of pain:

* by patients ≥4 years on the Wong-Baker scale, rated of 0–5 (0 = no pain; 5 = worst possible pain),

* by parents on the Visual Analogue scale, rated of 0–10 (0 = no pain and discomfort; 10 = worst pain and discomfort) and

* by nurses on the Behavioral Scale Face Legs Activity Cry Consolability, rated of 0–10 (0 = no discomfort and 10 = worst discomfort).

For the ST, pain and discomfort were rated just before swallowing the capsule, during swallowing of the capsule, immediately after swallowing it; just

before removing the capsule, during removal of capsule and immediately after removal of capsule.

## Tuberculosis diagnostic techniques

All bacteriological samples were decontaminated with N-acetyl-L-cysteine and sodium hydroxide and centrifuged; stool samples were previously prepared by dilution in sterile water. Centrifuged pellets were resuspended in phosphate buffer. Direct exam was performed using the Ziehl–Neelsen method for stool samples and fluorescent (auramine) microscopy from a drop of pellet suspension was performed for all other samples. Sample pellet suspensions were inoculated in Lowenstein–Jensen slants. Isolates were identified as *Mycobacterium tuberculosis* complex by Ziehl–Neelsen staining and biochemical method. The Xpert assay was performed on 1.0 ml of fresh sample or decontaminated pellet if the sample volume was not sufficient, following the manufacturer's recommendations. Stool samples were previously processed by emulsification of 0.5 g of material in Sheather's solution (28% sucrose in distilled water), filtered through funnel gauze and centrifuged.

The diagnosis of active TB was made in any child with:

* clinical signs suggestive of TB considering the medical history,

* and/or a positive culture for *M. tuberculosis*,

* and/or a positive microscopic examination,

* and/or a positive GeneXpert®,

* and/or radiographic evidence suggestive of TB considering the medical history and

* and/or ultrasound suggestive of TB considering the medical history.

## Sample size

The sample size of PAANTHER 01 study was initially determined to allow random split sample development and validation. With an expected sensitivity and specificity of 90% for the model and a precision of 15% and expecting that two-thirds of children would be diagnosed with tuberculosis, we estimated that we would need 210 children (40 were planned in Burkina Faso) in the development group and an identical number in the validation group. The study
scientific committee secondarily approved the devel-
opment of the model on the full data set and
approved internal validation by using bootstrap
resampling.

Data analysis

Data were analyzed by a microcomputer equipped
with the French version 3.1 of the Epi-Data software
and Microsoft Office 2016.

Clinical Trials Registration: the study was approved by national ethic committee of Burkina Faso: number 2011-8-47 of 03 August 2011. The ANRS 12229 PAANTHER 01 Study is registered at ClinicalTrials.gov (NCT01331811).

RESULTS

During the study period, 63 HIV-infected children
and suspected of having TB were included. Baseline
characteristics are shown in Table 1.

Sixty-two hemogram have been made. One child
died before. Half of the patients (31/62) were an-
emic. Mean CD4 in our study population was
743.40 ± 650.61/µl. Of the 63 children at inclusion,
42 had a detectable viral load and 52.38% were on
anti-retroviral (ARVs). Basis of anamnestic, clinical
and paraclinical arguments, the frequency of TB was
49.2% (31/63): 22 TB pulmonary and 9 extra pul-
monary. Of the 31 cases of TB, bacteriological con-
firmation was made on 21 samples, taken from 12
patients (each child having at least 5 samples, some
had several positive samples). One patient was posi-
tive on both microscopy and Xpert® MTB/RIF. And
two positive patients for both culture and Xpert®
MTB/RIF.

Feasibility

GA was performed in seven children <4 years old,
32 aged 4–9 years (1 patient had a very abundant
epistaxis, contraindicating GA or NPA) and 5 aged
10–13 years unable to produce enough sputum. In
total, 95 sample of GA were obtained. During the
procedure, adverse events occurred in 6/7 (85.71%)
children aged below 4 years old at first GA, and in all
(100%) children at second and third GA. There
were essentially: cough (20/21 GA), sneeze (14/21
GA), traumatism and nosebleed (4/21 GA) and
vomiting (1/21 GA).

For children aged 4–9 years, adverse events occurred in 29/32 (90.62%) at first GA and 27/32 (84.37%) at second GA: cough (48/64 GA), sneeze (36/64 GA), traumatism and nosebleed (6/64 GA), nausea (11/64 GA) and vomiting (5/64 GA).

NPA was performed in 62/63 patients. Adverse
events occurred in 53 patients: 6 in under 4 years age
group, 29 in the 4–9 aged group and 18 in the over
10 aged group. The nature and frequency of adverse
events occurring during NPA by age group of
patients are shown in Table 2. Twenty-two (84.62%)
malnourished patients (Z score< −2) had adverse
events during NPA. There was no significant associ-
ation between nutritional status (Z score< −2) and
the feasibility of NPA (Fisher’s exact = 1.000); nor
between anemia and the feasibility of NPA (Fisher’s
exact = 0.081).

In terms of feasibility of the ST, 55/56 (98.21%) children were able to swallow the capsule: 32 (96.96%) aged 4–9 years and 23 (100%) aged 10–13 years. Indeed, one child aged 4–9 years, was unable to swallow the capsule of the ST. At the end of the study, ST had been successfully completed in 53/55 (96.36%) children: one patient aged 4–9 year has vomited the capsule 15 min after swallowing; one child aged 10–13 years has vomited the capsule a few minutes before the scheduled time of withdrawal.

Adverse events occurred in eight children in
4–9 years of age and in three patients in 10–13 years
of age. These were nausea and vomiting. Six mal-
nourished children (27.27%) had adverse events dur-
ing ST. There was no significant association between
the feasibility of ST and nutritional status (Z score<
-2) (Fisher's exact = 0.508), WHO stage (Fisher's
exact = 0.551) and anemia (Fisher's exact = 0.328).

Eight children had no adverse events both at
NPA and ST.

Regarding stool collection, 3 of 63 patients had
not passed stool at the time of collection.

Tolerability

For GA, on behavioral scale, the highest pain score
was during collection: 7.51/10 for children <4 years
old and 6.4/10 for those 4–9 aged. On visual ana-
logue scale, the highest pain score were during
<table><caption>TABLE 1. Baseline characteristics</caption><thead><tr><th>Variables</th><th>Sub-group</th><th>Value (n = 63)</th></tr></thead><tbody><tr><td>Age (year), median (IQR)</td><td></td><td>8 (6–10)</td></tr><tr><td>&lt;4</td><td></td><td>7 (11.1%)</td></tr><tr><td>4–9</td><td></td><td>33 (52.38%)</td></tr><tr><td>10–13</td><td></td><td>23 (36.51%)</td></tr><tr><td>Sex</td><td>Female</td><td>33 (52.38%)</td></tr><tr><td>BCG vaccination</td><td>Received</td><td>51 (80.95%)</td></tr><tr><td></td><td>Not received</td><td>3 (4.76%)</td></tr><tr><td></td><td>Undocumented</td><td>9 (14.29%)</td></tr><tr><td>BCG scar</td><td>Present</td><td>45 (71.43%)</td></tr><tr><td></td><td>Absent</td><td>18 (28.57%)</td></tr><tr><td>History of TB more than 2 years</td><td>Yes</td><td>8 (12.7%)</td></tr><tr><td></td><td>No</td><td>55 (87.30%)</td></tr><tr><td>Type of previous TB</td><td>PTB+</td><td>1 (12.5%)</td></tr><tr><td></td><td>PTB-</td><td>3 (37.5%)</td></tr><tr><td></td><td>Extra pulmonary TB</td><td>2 (25%)</td></tr><tr><td></td><td>Undocumented</td><td>2 (25%)</td></tr><tr><td>TB exposure</td><td>Total</td><td>10 (15.87%)</td></tr><tr><td></td><td>At home</td><td>10 (100%)</td></tr><tr><td></td><td>Away from home</td><td>0</td></tr><tr><td>People exposed to TB at home</td><td>Mother</td><td>6 (60%)</td></tr><tr><td></td><td>Other person</td><td>4 (40%)<sup>a</sup></td></tr><tr><td>Malnutrition (WAZ score&lt; −2)</td><td></td><td>26 (41.27%)</td></tr><tr><td>WHO HIV clinical staging</td><td></td><td>43 (68.25%)</td></tr><tr><td>Stage 1</td><td></td><td>1 (1.52%)</td></tr><tr><td>Stage 2</td><td></td><td>14 (22.22%)</td></tr><tr><td>Stage 3</td><td></td><td>5 (7.94%)</td></tr><tr><td>Stage 4</td><td></td><td>31 (49.2%)</td></tr><tr><td>TB frequency</td><td></td><td>12 (38.7%)</td></tr><tr><td>% children confirmed TB to bacteriology</td><td></td><td></td></tr><tr><td>&lt;4 years</td><td></td><td>2</td></tr><tr><td>4–9 years</td><td></td><td>7</td></tr><tr><td>10–13 years</td><td></td><td>3</td></tr></tbody></table>

BCG, Bacille Calmette Guérin; TB, tuberculosis; PTB+, microscopic positive pulmonary tuberculosis; PTB-, microscopic negative pulmonary tuberculosis; WAZ score, weight for age Z score; WHO, World Health Organization; HIV, human immunodeficiency virus; IQR, inter-quartile interval.

collection too: 5.2/10 for those <4 years and 4.4/10 for 4–9 years old.

For NPA, and for all age groups, pain and discomfort scores assessed by children, nurses and parents were highest during the procedure, ranging from 5 to 6.36/10 (Fig. 1).

For ST, evaluated on all three scales, the average pain scores were highest when the capsule was swallowed and the string removed. They ranged from

0.15 to 1.28 for the 4–9 age group (Fig. 2) and 0 to 0.68 for the 10–13 age group.

## Results of diagnostic tests

In the 31 children diagnosed with TB, bacteriological confirmation was made on 21 samples, taken from 12 patients (each child having at least 5 samples, some had several positive samples). One patient was
<table><caption>TABLE 2. Adverse events during NPA by age group of patients</caption><thead><tr><th>Adverse events</th><th>&lt;4 years (n = 7)</th><th>4–9 years (n = 32)</th><th>10–13 years (n = 23)</th><th>Total</th></tr></thead><tbody><tr><td>Cough</td><td>6</td><td>26</td><td>16</td><td>48</td></tr><tr><td>Sneeze</td><td>5</td><td>20</td><td>13</td><td>38</td></tr><tr><td>Nausea</td><td>0</td><td>3</td><td>1</td><td>4</td></tr><tr><td>Vomiting</td><td>1</td><td>1</td><td>1</td><td>3</td></tr><tr><td>Trauma and nosebleed</td><td>3</td><td>3</td><td>2</td><td>8</td></tr><tr><td>Bradycardia</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Dyspnea</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Oxygen desaturation</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>Fig. 1. Assessment of tolerability of nasopharyngeal aspirate in children 4–9 years of age.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 2. Tolerability assessment of children aged 4–9 years old for the string test.</figcaption></figure>

positive on both microscopy and Xpert® MTB/RIF. And two positive patients for both culture and Xpert® MTB/RIF. There were 10/162 (6.17%)

standard samples and 11/177 (6.21%) alternative samples. Table 3 presents the detailed results of the microbiological tests.
<table><caption>TABLE 3. Detailed results of the microbiological tests</caption><thead><tr><th>Samples</th><th>Total</th><th>Positive microscopy</th><th>Positive culture</th><th>Positive GeneXpert®</th></tr></thead><tbody><tr><td>Standard samples</td><td>162</td><td>2</td><td>3</td><td>5</td></tr><tr><td>Gastric aspirate</td><td>95</td><td>2</td><td>3</td><td>3</td></tr><tr><td>Sputum</td><td>67</td><td>0</td><td>0</td><td>2</td></tr><tr><td>Alternative samples</td><td>177</td><td>0</td><td>4</td><td>7</td></tr><tr><td>NPA</td><td>62</td><td>0</td><td>2</td><td>4</td></tr><tr><td>String test</td><td>55</td><td>0</td><td>0</td><td>2</td></tr><tr><td>Stool</td><td>60</td><td>0</td><td>2</td><td>1</td></tr><tr><td>Total</td><td>339</td><td>2</td><td>7</td><td>12</td></tr></tbody></table>

# DISCUSSION

## Feasibility and tolerability

Of the variety of methods for obtaining clinical specimens for bacteriological confirmation of pediatric tuberculosis, GA to obtain swallowed bronchial secretions is the most commonly used [16–18]. In our study, GA had poor tolerability for all age group. Pain was highest during procedure. This corroborates the data from the literature on the unpleasant nature of GA [5–7, 19].

NPA can be done at any time and does not require overnight fasting, although it is best done 2 h after the last meal to decrease the risk of vomiting. NPA has been used successfully in many reports [20–25]. But data on the acceptability of the methodology are lacking in these studies. In our study, NPA was unpleasant. It had a poor feasibility: adverse events occurred in 85.48% (53/62) of cases.

The mean score of discomfort rated by children, parents and nurses was highest during the procedure. The other stages of the procedure were much better tolerated by children.

For the ST, among patients aged 4–13 years, 98.21% (55/56) had swallowed the capsule. This rate is higher than that of other authors: Chow, et al. (90.47%) [16], Nansumba, et al. (83.94%) [26], Marcy, et al. (79%) [4] and Tafur, et al. (34%) [19]. Like these authors, we found that older children (age ≥ 10 years) were generally able to swallow the capsule and successfully complete the test, compared with younger children.

In terms of feasibility of ST, one potential explanation for differential ST success across settings relates to pill taking familiarity [19]. For example,

one-third of children who participated in the study conducted by Nansumba, et al. [26] were living with HIV and therefore may have been more accustomed to swallowing pills. In our study, 52.38% were on ARV.

The results of this study indicate that the ST is well tolerated by the majority of children over 4 years old. The brief moments of capsule swallowing and string retrieval were although less well tolerated. However, it remained very low, below 2 of 10. Our findings are consistent with previous reports: Chow, et al. [16] and Nansumba's, et al. [26] with a maximum pain and discomfort score during insertion and removal of the ST, but did not exceed 3 of 10.

In Tafur's study, ST was better tolerated by all age groups of children compared with GA [19].

During ST, there were very few adverse events, and none was serious. For Chow, et al. [16], adverse events during ST removal were tickling, pricking or scraping sensation in the throat, sensation of being choked and could not breathe, nausea and feeling afraid.

Capsule size is certainly a serious limiting factor, especially in children <4 years. Smaller capsules and additional interventions to facilitate pill swallowing, such as behavioral therapies, flavored throat spray, verbal instructions, specialized pill cup and head posture training [27] could improve ST feasibility. This is particularly important in countries with a high HIV burden and where the median age of children with tuberculosis is <4 years [28]. In Chow's study, 30.30% of children used honey and water to swallow the ST capsule [16].

As for stool, 60/63 stool samples could be collected. Stool samples are used to collect bacilli from respiratory sources and their use in the diagnosis of
tuberculosis has been demonstrated in both culture and other tests [18, 29, 30]. For Wolf, et al. [30] diagnosing tuberculosis in stool in children is initial and rapid test, especially if sensitivity can be increased. So, patients who test positive may avoid multiple invasive procedures.

## Results of diagnostic tests on different bacteriological samples

In our study, the contribution of alternative samples to the diagnosis of tuberculosis was essentially identical to that of standard samples: *M. tuberculosis* was detected in 11 alternative samples (in 10 patients) and 10 standard samples (in 7 patients). Alternative samples could therefore replace or supplement standard samples when they are difficult to obtain.

The bacteriological confirmation rate of TB was higher in our study than in others: 10.2% [26], 38% [19] and 0% [16]; probably due to the multiplicity of bacteriological samples in our study. Studies that evaluated SI, GA or NPA in children report an average increase of 20% of TB detection yield when using two specimens compared with one specimen [18]. This is consistent with Healter finding [22]. Thus, a combination of different specimen collection methods might increase the bacteriological yield toward the diagnosis of TB in children and offer a possibility of a 1-day TB diagnosis in children [26].

## Limitations of the study

Our study had some limitations. A small size of our sample limits us from generalization of our results.

## CONCLUSION

This work has clearly established that the ST methodology is well tolerated by children as young as 4 years of age and is highly acceptable to the children, their parents and their attending nursing staff. Stool could be useful in patients with difficulty in expectorating out sputum. NPA has a poor feasibility and tolerability in children, although its ability to screen for *M. tuberculosis* has been proven in the literature.

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