microorganisms

Review
Pulmonary Tuberculosis in Children: A Forgotten Disease?
Umberto Pelosi 1 , Roberta Pintus 2, * , Salvatore Savasta 3 and Vassilios Fanos 2

1 Pediatric Unit, Santa Barbara Hospital, 09016 Iglesias, Italy

2 Neonatal Intensive Care Unit, Department of Surgical Sciences, University of Cagliari, AOU Cagliari,
09124 Cagliari, Italy
3 Department of Pediatrics and Rare Diseases, Ospedale Microcitemico Antonio Cao, University of Cagliari,
09124 Cagliari, Italy
* Correspondence: [email protected]

Abstract: Even today, tuberculosis in childhood is a disease that is often undiagnosed and under-
treated. In the absence of therapy with antituberculosis drugs, children in the first years of life have a
high degree of severe forms and mortality. In these children, symptoms are often not very specific
and can easily be confused with other diseases of bacterial, viral or fungal etiology, making diagnosis
more difficult. Nevertheless, the introduction of new diagnostic techniques has allowed a more rapid
identification of the infection. Indeed, Interferon gamma release assay (IGRA) is preferred to the
Mantoux, albeit with obvious limitations in children aged <2 years. While the Xpert Mtb/RIF Ultra
test is recommended as an initial diagnostic investigation of the gastric aspirate and/or stools in
children with signs and symptoms of pulmonary tuberculosis. The drugs used in the treatment of
susceptible and resistant TB are the same as those used in adults but doses and combinations are
different in the pediatric age. In children, brief therapy is preferable in both the latent infection and
the active disease, as a significant reduction in side effects is obtained.

Keywords: children; pathogenesis; diagnosis; skin tests; Xpert MtB/Rif Ultra; therapy; prevention

1. Introduction
Citation: Pelosi, U.; Pintus, R.;
To date, about 1.5 million children contract tuberculosis (TB) every year. They represent
Savasta, S.; Fanos, V. Pulmonary
1.1% of the entire affected population, of these in 2020, only 36.5% were notified to the
Tuberculosis in Children: A Forgotten
competent authorities. TB is currently considered one of the 10 causes of death in childhood
Disease? Microorganisms 2023, 11,
1722. https://doi.org/10.3390/
as stated in the WHO Global Tuberculosis Report. Furthermore, this document stated that,
microorganisms11071722
in 2020, TB was responsible for 16% of global deaths in children. The main cause of these
deaths is above all the reduction, particularly in the most endemic countries, of access to
Academic Editor: Antonella health facilities and the adherence to therapeutic protocols. In Italy in 2020, 2287 cases of TB
d’Arminio Monforte
were recorded, 1.8% aged <5 years, 2.8% aged 5–14 and 15.1% aged 15–24 [1–3]. Currently,
Received: 15 March 2023 it is considered one of the 10 causes of death in childhood. In the course of the pandemic of
Revised: 20 June 2023 SARS-CoV-2, in 2020, a sharp drop occurred in access to diagnosis and therapy services
Accepted: 26 June 2023 for children aged 5–15 years (63%) and a clear reduction in prophylaxis for tuberculosis
Published: 30 June 2023 infection in children < 5 years of age (72%). The reduction in access to hospital facilities
has consequently led to a significant increase in new cases and deaths in the following
years [4–6]. Furthermore, children are more vulnerable to tuberculosis infection because
of their immunological immaturity characterized by an altered innate immunity in the
Copyright: © 2023 by the authors.
lungs, due to a reduced recruitment of macrophages to the site of infection and a decreased
Licensee MDPI, Basel, Switzerland.
production of IL-12, which is essential for the initial polarization of the Th1 lymphocytes,
This article is an open access article
which, in turn, are important to maintain an efficient transcription of IFN-γ [7–9].
distributed under the terms and
Moreover, children aged <5 years are more exposed to the infection and to the most
conditions of the Creative Commons
severe forms of the disease, indeed about, in pediatric age, 80% of TB deaths in children
Attribution (CC BY) license (https://
occur mainly in this age group. Sometimes these children have silent forms of TB with mild
creativecommons.org/licenses/by/
4.0/).
non-specific symptoms and do not develop the disease. In these cases, there is a 10% risk of

Microorganisms 2023, 11, 1722. https://doi.org/10.3390/microorganisms11071722 https://www.mdpi.com/journal/microorganisms

 mild non-specific symptoms and do not develop the disease. In these cases, there is a 10
risk of reactivation in adulthood. In addition, in children, the disease has som
peculiarities that differentiate it from the adult forms [10–12]. These features a
paucibacillary; a low incidence of cavitary forms; an increased frequency in evolution
Microorganisms 2023, 11, 1722 the infection into the disease; a more rapid and severe course in children < 22ofyears; 16 a hig
incidence of extrapulmonary forms; in 20–60% of cases it can be symptomatic; radiolog
is often of little significance; and diagnostic difficulties. Some of these are discussed in t
reactivation in adulthood. In addition, in children, the disease has some peculiarities that
following paragraphs.
differentiate it from the adult forms [10–12]. These features are paucibacillary; a low inci-
dence of cavitary forms; an increased frequency in evolution of the infection into the disease;
2. Pathogenesis
a more rapid and severe course in children < 2 years; a high incidence of extrapulmonary
forms;Mycobacterium
in 20–60% of casestuberculosis (MTB), an intracellular
it can be symptomatic; pathogen,
radiology is often of littleissignificance;
transmitted from
sickdiagnostic
and adult through exhaled
difficulties. Someair and are
of these thediscussed
emissionin of
thedroplets
followingof infected secretions v
paragraphs.
sneezing and/or coughing. These droplets are particles measuring 1–5 microns that c
2. Pathogenesis
transport 2–3 bacteria; the infection charge is around 10–150 bacteria; coughing, singin
and talking for 5tuberculosis
Mycobacterium (MTB), an3000
min can produce intracellular
dropletspathogen,
[13,14].isThis
transmitted from a sick has a c
microorganism
adult through exhaled air and the emission of droplets of infected secretions via sneezing
wall that is rich in lipids, which confers some particular characteristics: acid resistanc
and/or coughing. These droplets are particles measuring 1–5 microns that can transport
slow growth and the possibility of surviving inside macrophages [15]. Moreover, a chi
2–3 bacteria; the infection charge is around 10–150 bacteria; coughing, singing and talking
is hardly
for able
5 min can to infect
produce 3000another
dropletschild. Indeed,
[13,14]. in children they
This microorganism has are
a cellthe
wallcavitary forms exce
that is rich
in lipids, which confers some particular characteristics: acid resistance, slow growth and infectio
in those with HIV or with genetic modifications in the immune response. The
begins
the when of
possibility thesurviving
MTB enters
insidethe upper airways
macrophages via inhalation
[15]. Moreover, a childwhere 90%
is hardly is expelled
able to b
infect another child.
the mucociliary Indeed,and
system in children
by the they are the
immune cavitary
cells forms
present inexcept in those
the lamina with [16,1
propria
HIV
After or bypassing
with geneticthemodifications
upper airwayin thedefense
immune response.
system, MTBThe infection
enters thebegins whenusually
lungs the in t
MTB enters the upper airways via inhalation where 90% is expelled by the mucociliary
middle lobe and lower lobes reaching the alveoli (<10%) where it encounters alveol
system and by the immune cells present in the lamina propria [16,17]. After bypassing the
macrophages, dendritic cells and neutrophils, which binds to their surface [18–20].
upper airway defense system, MTB enters the lungs usually in the middle lobe and lower
However,
lobes reaching the this exposure
alveoli leadsit encounters
(<10%) where to two events:
alveolarthe elimination
macrophages, of thecells
dendritic germ or
persistence, and about 10% of infected
and neutrophils, which binds to their surface [18–20].people have a chance of developing the acti
disease [21,22].
However, These
this events
exposure aretoillustrated
leads two events:intheFigure 1.
elimination of the germ or its per-
sistence, and about 10% of infected people have a chance of developing the active dis-
ease [21,22]. These events are illustrated in Figure 1.

Figure 1. Chronological events after MTB inhalation.

Figure 1. Chronological events after MTB inhalation.
An inadequate immunological response or an age < 2 years facilitate continuous
mycobacterium replication and its progression to lung disease or its dissemination to
other An inadequate
districts (Figure 1).immunological response
On the other hand, childrenoraged
an 5–10
age years
< 2 years
have afacilitate continuo
low risk of
mycobacterium
disease progression,replication and its
while adolescents progression
have todisease
a high risk of lung disease or [23].
reactivation its dissemination
Table 1
other districts (Figure 1). On the other hand, children
illustrates the risk of progression of TB according to age (Table 1).aged 5–10 years have a low risk
disease progression,
Moreover, there arewhile adolescents
several factors that have
favor a high
the risk offrom
transition disease reactivation
infection to disease[23]. Tab
and its severity, summarized in Table 2 [24].
1 illustrates the risk of progression of TB according to age (Table 1).
From an immunological point of view, both innate and cell-mediated immunity are
fundamental to contain the TB infection and their interplay contributes to the clinical
presentations of TB [25,26]. The inhaled MTB, in immunocompetent subjects, binds to
the surface of neutrophils and alveolar macrophages [27,28]. Neutrophil phagocyte MTBs
kill them following the release of lysosomal enzymes and the production of reactive
oxygen intermediates (ROI), human neutrophil peptides (HNPs), myeloperoxidase (MPO),
neutrophil serine protease (NSP) and lactoferrin (LA) [29,30]. Macrophages recognize MTB
Microorganisms 2023, 11, 1722 3 of 16

by PPR3 membrane receptors which, when stimulated, via intracellular messengers activate
transcription, leading to the production of proinflammatory cytokines and chemokines.
In turn, this leads to an increased vascular permeability, causing the recruitment of other
inflammatory cells, in particular dendritic cells. Thus, phagocytosis is activated through
various mechanisms: (a) recognition of the mannose residues of the MTB by the receptors of
the macrophage where the lipoarabinomannan present on the surface binds, (b) activation
of the C3 proteins of the complement present in the alveolar space, which are recognized
by the complement receptors present on the surface of macrophages [31–33].

Table 1. Risk of progression of TB according to age.

Age Risk (%)

<12 months 40–40
1–2 years 25
School age 5–10
Adolescents 10–15
Adults 5–10

Table 2. Risk factors for tuberculosis in children after the infection.

Risk Factors for the Transition from Infection to Disease TB in Children

• Presence of chronic cough
• Extrapulmonary tuberculosis
• Poor use of health facilities
• First non-specialist physician examination, such as family pediatrician with no experience
with TB in children
• Immigration history
• Low socio-cultural level

Thus, MTB is phagocytosed inside the vacuoles and following the secretion of some
cytokines and chemokines (IFN-γ, TNF-α, IL-1β, IL-6 and IL-23) is killed or limited in
its growth [34]. Furthermore, most of the macrophages control the infection by rapidly
activating some antimicrobial mechanisms: (a) fusion of phagosomes with lysosomes
(formation of the phago-lysosomal complex), (b) autophagy, and (c) oxidative stress [33–35].
Nevertheless, bronchoalveolar cell responses featured in TB patients consist of lymphocytic
alveolitis and immature macrophage alveolitis. This initial response involving innate
immunity is sufficient to eliminate MBT.
However, it tries to survive inside the host by inhibiting: (a) the fusion of phagosomes
with lysosomes, (b) the maturation of macrophage phagocytosis by suppressing phagosome
acidification and (c) the production in macrophages of ROS by catalase-peroxidase (Kat
G), by reducing the action of oxidative stress [36–39]. Then, it is able to replicate inside the
macrophages and to invade the pulmonary interstitium.
For their part, macrophages and dendritic cells, resident in the pulmonary interstitium,
phagocytize MTB and migrate to the lymph nodes where they present MTB antigens to T
cells that differentiate into CD4+ and CD8+ [40,41]. Then, the CD4 and CD8 cells return to
the lungs thanks to the chemokines produced at the site of infection. Here, these activated
T cells bind to the MHC/antigen complexes on the surface of the infected macrophages
and produce a range of cytokines, including IFNγ, leading to the further activation of
macrophages that kill MTBs that escaped the initial phagocytosis [39,42]. Moreover, TNF-α
produced by macrophages, T cells, dendritic cells, fibroblasts and endothelial cells plays a
key role in the formation of the granuloma at the site of the infection [43,44].
Indeed, the accumulation of lymphocytes, macrophages, dendritic cells and epithe-
lioid cells at the site of the infection determines the formation of the granuloma within
which the MTB is contained and where it can persist for several decades. In healthy close
Microorganisms 2023, 11, 1722 4 of 16

contacts of TB patients and during active pulmonary TB, the immune response is confined
to the lung and characterized by an exuberant Th1 lymphocyte response, which is counter-
acted by local suppressive immune mechanisms, as suggested by the recent evidence [45].
Generally, a patient with a primary MBT infection is asymptomatic and undergoes clinical
but not biological recovery, remaining infected with quiescent mycobacteria. This clinical
quiescence often persists for the entire life of the individual. In a smaller percentage of
cases, subjects with tuberculosis infection may immediately manifest the active disease
(primary tuberculosis) or undergo a clinical reactivation of the infection during their life
(secondary tuberculosis), due to the lack of development or maintenance of an effective
immune response.

3. The Role of Microbiota

In the past, the scientific community believed that the lungs of healthy subjects were
germ-free and that they could only be colonized in cases of lung pathology. This miscon-
ception resulted in the exclusion of the lung from the Microbiome Human Project [46].
Only starting in 2010, an increasing number of studies have shown, through new DNA
and RNA investigation techniques, that the lungs of healthy subjects are not germ-free
but are colonized by bacteria, viruses and fungi [47,48]. These new techniques lead to
the identification of different species of bacteria: (1) at the phylum level: Firmicutes, Bac-
teroides and Proteobacteria, (2) at the genus level: Veillonella, Prevotella, Fusobacteria and
Streptococcus, with the presence of small quantities of pathogens, such as Haemophilus and
(3) fungi: Aspergillus, Cladosporium, Penicillum and Eurotium [48]. The lungs are colonized by
different species in their different the anatomical region (bronchi, bronchioles and alveoli)
thus, the constituents of the lung microbiota depends on several factors, some of which are
particularly important, such as (1) microbial immigration (micro-aspiration, inhalation of
microorganisms, direct mucosal dispersion), (2) microbial elimination (cough, mucociliary
clearance, innate and adaptive immunity) and (3) regional growth conditions (nutritional
availability, temperature, O2-tension, local microbial competition, concentration and ac-
tivity of inflammatory cells) [49]. The equilibrium of these elements is essential for the
composition of the lung microbiota in healthy subjects. The local factors in a healthy subject
determines the adverse growth and a multiplication environment for the microorganisms.
On the other hand, when the environment shifts, the reproduction of pathogens and the sub-
sequent occurrence of pathologies, which can often become chronic, is favored. Moreover,
the microbiota present in the human body produces metabolites capable of modulating the
host’s immune response. Even microbial communities present at distal sites of the body
play an important role in respiratory health.
Furthermore, a theory has been advanced, namely that some essential risk factors for
tuberculosis (HIV, malnutrition, diabetes, alcohol, smoking and pollution) interact with the
host through the microbiota [50,51].
In addition, recent scientific evidence proves the contribution of the microbiota in
TB pathogenesis, therapy response, clinical outcomes, and post-treatment outcomes [52].
This role is underpinned by complex multifactorial interactions between the pathogen, the
commensal flora and the guest.
In particular, some peculiar aspects have been identified:
(1) Susceptibility to infection and progression to active TB is modified by intestinal
Helicobacter co-infection;
(2) Airborne Mycobacterium tuberculosis infection modifies the intestinal microbiota;
(3) Anaerobes present in the lungs and coming from the oral cavity by aspiration produce
metabolites that reduce lung immunity and predict progression;
(4) The increased susceptibility to reinfection of patients who have been previously
treated for TB is probably due to the depletion of antigenic epitopes for T cells in the
commensal intestinal flora (non-tuberculous mycobacteria);
(5) The prolonged antibiotic treatment necessary for tuberculosis has long-term detrimen-
tal effects on the microbiome.
Microorganisms 2023, 11, 1722 5 of 16

Moreover, numerous evidences suggest that the gut microbiota modulates the host
immune response against MTB and that this mechanism could constitute a potential
therapeutic target [52].
Additionally, subjects affected by TB present a greater abundance of Streptococcus,
Gramulicatella and Pseudomonas. While Prevotella, Leptotrichia, Treponema, Catonella and
Coprococcus are less represented in subjects with TB than in healthy controls.
Moreover, subjects with recurrent TB show a reduced frequency of some genera, such
as Bulleidia and Atopobium compared to subjects with new onset TB.
Finally, it has recently been hypothesized that the microbiome through the gut/lung
axis plays an important role in granuloma formation, promoting the Th1 and Th17 re-
sponse [52].

Effects of Therapy on the Microbiota

Several studies have demonstrated that the antibiotic administration is responsible for
the alterations in both the intestinal and pulmonary microbiota [53]. The risk is inversely
related to the age of exposure and to the amount and classes of antibiotics and determines
important modifications in the immune response of the host against the pathogens, favoring
the progression and severity of the disease [54]. As observed in other diseases, the adminis-
tration of these drugs is responsible for the modifications in the intestinal and pulmonary
microbiota [55]. While the effects on the intestinal microbiota are well documented, at
present, there are few studies investigating the pulmonary dysbiosis [56]. This scarceness of
information is closely correlated with the variety of pulmonary conditions that can arise in
the course of lung injury (fibrosis, cavitations, broncho-stenosis, bronchiectasis, alteration of
the parenchyma) in relation to age, diffusion and severity of the disease. The first particular
evidence present in most of the investigations is that therapy with isoniazid, rifampicin,
pyrazinamide and ethambutol (HRZE) has minimal effects on the diversity of the phyla
of the intestinal microbiota but determines evident alterations on their distribution and
quantity [57].
Animal studies and studies performed on humans with tuberculosis treated with
HRZE show an increase in the genus of the phyla Bacteroides and Proteobacteria and a sig-
nificant reduction in the phylum Firmicutes (Clostridiales) [58,59]. Another particular aspect
of the alterations of the microbiota related to the therapy administration is the persistence
of dysbiosis for 1 to 3 years after its suspension (chronic effects). Markers of persistent
dysbiosis are the reduction in Bacteroides, Firmicutes (Clostridiales, Ruminococcus, Faecal-
ibacterium) and an increase in Actinobacteria and Proteobacteria (Escherichia, Salmonella,
Yersinia, Helicobacter) [60].
It has been speculated that the acute and chronic dysbiosis that occurs during therapy
may be responsible for three possible conditions:
(1) Alteration of the host immune response that can influence the course and severity
of the disease. Dysbiosis significantly reduces the immune response against MTB
and leads to an increase in inflammation in the course of the disease. The reduction
in Bacteroides and Firmicutes, in particular of Ruminococcus and Coprococcus, which
regulate the expression of IL-1 and IFN-γ and of Bifidobacterium, which induces
a reduction in the activity of Th17 cells, are responsible for these alterations [61].
Finally, another significant effect of dysbiosis is the decrease in the bactericidal effect
of macrophages due to a decrease in their autophagy [62,63];
(2) Modification of the therapy response: there is no significant evidence in the literature
to confirm the importance of dysbiosis on the efficacy of drugs, although it is hypoth-
esized that the modifications affecting the intestinal mucosa and its barrier function
may cause a reduced absorption and metabolism of the drugs themselves [64];
(3) An increased risk of reinfection: the persistence of dysbiosis, years after the suspension
of therapy, seems to be responsible for an increased risk of reinfection [65]. It is
commonly believed that the lower resistance of the host against MTB is related to an
Microorganisms 2023, 11, 1722 6 of 16

individual profile of the host microbiota, its profile before the disease or individually
modified by therapy, which interacts with specific epitopes of the pathogen.

4. Vitamin D and Tuberculosis

Numerous studies have shown that vitamin D is capable, as occurs in other infec-
tious processes, of modulating the immune response against MTB, in particular through
the activation of No/ROS, which determines the inhibition of MTB growth in infected
macrophages and LL-37, which modulates the production of proinflammatory cytokines.
In detail, there is an increase in IL-10 and IL-12 and a decrease in TNF-α, with consequent
reduction in the inflammatory state and tissue damage induced by MTB [14,66].

5. Diagnosis
Nowadays, diagnosis in children is often difficult, as the signs and symptoms, espe-
cially in the first years of life, can be confused with other pathologies.
The diagnosis is based on 1. Anamnesis, 2. Clinical criteria, 3. Skin Test 4. New
diagnostic tests 5. Bacteriological tests and 6. Radiological investigations.

5.1. Anamnesis
The medical history of a contact with known cases of TB allows to suspect infection
or disease. In children and in particular in those aged <2 years, the symptoms are often
not very specific and can easily be confused with other diseases of bacterial, viral or fungal
etiology, making the diagnosis more difficult [9,67]. Signs and symptoms that can guide
the diagnosis are reported in Table 3.

Table 3. Signs and symptoms of tuberculosis in children.

Signs and Symptoms of Tuberculosis in Children

• persistent cough > 4 weeks
• fever (often low-grade and intermittent)
• night sweats
• weight loss and anorexia
• general malaise and asthenia
• hemoptysis
• chest pain (if there are subpleural lesions)
• respiratory distress
• pneumonia resistant to antibiotic therapy

The simultaneity of some symptoms, such as persistent cough, listlessness and weight
loss, have a predictive value in 80% of children with pulmonary TB [11,68].

5.2. Clinical Criteria

Symptoms vary according to the age of the child and the affected site. The pulmonary
localization appears to be the most frequent (71.8%), while the extra-pulmonary localiza-
tion is present in about 20–30% of cases and includes the localization of the infection in
different districts: central nervous system (meninges, parenchyma and spinal cord) [69],
lymph nodes (lymphadenitis present in 30–40% of cases in the latero-cervical, supraclav-
icular, axillary and mediastinal, abdominal) [70], skeleton (spondylitis, osteomyelitis and
arthritis) [71,72], genitourinary system (present in 3% of cases, indicator of disease reactiva-
tion) [73], kidney (presence of pyuria in sterile urine) [74], gastrointestinal system [75] and
skin (scrofuloderma shown in Figure 2) [76].
 cord) [69], lymph nodes (lymphadenitis present in 30–40% of cases in the late
supraclavicular, axillary and mediastinal, abdominal) [70], skeleton (
osteomyelitis and arthritis) [71,72], genitourinary system (present in 3% of case
Microorganisms 2023, 11, 1722 of disease reactivation) [73], kidney (presence of pyuria in7 of sterile16 u
gastrointestinal system [75] and skin (scrofuloderma shown in Figure 2) [76].

Figure
Figure 2. Scrofuloderma.
2. Scrofuloderma.

5.3. Skin Tests

5.3.The
Skin Tests
conventional skin test (Mantoux) is certainly the oldest test that has allowed both
diagnostic
Theand epidemiological
conventional screening
skin [77,78]. Theisperforming
test (Mantoux) certainlyisthe
simple andtest
oldest requires
that has al
only minimal attention from the operator. Positivity appears 2–10 weeks after the beginning
diagnostic and epidemiological screening [77,78]. The performing is simple a
of the infection and is considered positive in relation to the size of the wheal. Positive
only minimal
Mantoux Definition attention
is described from
in Tablethe
4. operator. Positivity appears 2–10 week
beginning of the infection and is considered positive in relation to the size of
Table 4. Positive Mantoux definition in children.
Positive Mantoux Definition is described in Table 4.
Positive Mantoux Definition in Children
Table 4. Positive Mantoux definition
Children in children.
in close contact with a person with a known or suspected
tuberculosis infection
Children with
Positive Mantoux suspected tuberculosis
Definition in Children disease
Reaction ≥ 5 mm • Chest X-ray consistent with disease
Children
•
in close contact with a person with a known or suspec
Clinical signs of tuberculosis
tuberculosis infection
Children receiving immunosuppressive therapy or with congenital or
acquired immunodeficiency
Children with suspected tuberculosis disease
Reaction ≥ 5 mm • Children at increased risk of disseminated form:
Chest X-ray consistent with disease
• age < 4 years
• •Clinical signs
with risky of tuberculosis
clinical conditions (lymphomas, diabetes,
Childrenmalnutrition,
receivingetc.) immunosuppressive therapy or with conge
Reaction ≥ 10 mm Children with increased risk of environmental exposure:
acquired immunodeficiency
• Born in, or with parents coming from, highly endemic areas
Children
• at increased
Frequently exposedrisk of disseminated
to adults form:
with HIV, drug addicts, inmates
• •age < 4 years
Travel and exposure to regions of the world with high prevalence
Reaction ≥ 15 mm• with risky
Children clinical
≥ 4 years without conditions
risk factors (lymphomas, diabetes, maln
etc.)
Reaction ≥ 10 mm Nevertheless, the tuberculin test has some disadvantages that can make diagnosis
Children with increased risk of environmental exposure:
difficult. These disadvantages are shown in Table 5.
• Born in, or with parents coming from, highly endemic are
The main problem of the intra-dermal reaction is that it does not allow to distinguish
between infection and• disease
Frequently exposed
and between to adults
vaccinated with HIV,
and infected [79]. drug addicts, inm
Nonetheless,
three new tests have • recently
Travelbeenand exposure
developed for the to regions
diagnosis of the world
of MTB: with high pre
(a) Diaskintest,
(b) C-Tb skin test and (c) Ec-test. These tests are based on the presence of two MBT antigens:
Reaction ≥ 15 mm Children ≥ 4 years without risk factors
CFP-10 and ESAT-6. They have the advantage over the traditional skin test of differentiating
between positives from infection and positives from vaccination [80,81].
Nevertheless, the tuberculin test has some disadvantages that can mak
difficult. These disadvantages are shown in Table 5.
Microorganisms 2023, 11, 1722 8 of 16

Table 5. Factors that may make the evaluation of Mantoux difficult.

Factor That Can Affect the Mantoux Evaluation

• BCG vaccination
Positive • Non-tuberculous mycobacteria (NTM)
• measles vaccination
Negative • temporary anergy (2–12 weeks after infection), previous
administration of Corticosteroids or immunosuppressants

5.4. New Diagnostic Tests

Interferon-gamma release assay (IGRA) tests have been widely used in the last
decade [82]. These are based on the measurement of the IFN-y produced by T-lymphocytes,
following stimulation with two highly specific MBT antigens: ESAT-6 and CFP-10, which
are absent in M.bovis and the BCG [10,83]. Two second generation tests are currently on
the market: the Quantiferon-TB Plus and the T-SPOT.TB, which differ from the previous
ones in that they use new antigens which are able to stimulate both CD4 and CD8+ [84–86].
They have some advantages over the traditional skin test (summarized in Table 6).

Table 6. Comparison between Mantoux and IGRA.

Mantoux IGRA
Recommended age All >2 years
Cross reactivity with BCG YES NO
Distinction between infection and disease NO NO
Cross reactrivity with non tubercular Mb YES Not Much
Operator Dependent Not dependent
Cost Low High

These tests also do not allow to distinguish the latent infection from the disease, such
as the Mantoux test, and they are not recommended in children in the first years of life as
they have a low sensitivity in this age group [87–89].
Recently, new diagnostic tests based on the rapid determination of MTB DNA in the
sample (NAAT nucleic acid amplification test and Xpert Mtb/RIF Ultra test). While the
LF-LAM test (Lateral Flow Urine Lipoarabinomannan) is based on the identification of the
liposaccharide antigen LAM present on the cell wall of MTB [35].
Among these tests, the most recommended [6] are the Xpert Mtb/RIF Ultra and the
LF-LAM. The first is recommended as an initial test in pediatric ages in cases of pulmonary
tuberculosis. It can be performed on various samples: feces (97–100% specificity and
32–90% sensitivity) and gastric aspirate (94% specificity and sensitivity of 71.5%). The Xpert
Mtb/RIF Ultra assay appears to be useful, in addition to diagnosis, for the identification
in a single assay of resistance-associated mutations for rifampin, isoniazid, amikacin and
capreomycin [90–93]. While the LF-LAM test (Lateral Flow Urine Li-poarabinomannan) is
indicated as an additional test in HIV positive children, presenting signs and symptoms of
pulmonary tuberculosis and in those who are HIV positive without symptoms with a CD4
cell count < 100/mm3 [6,8].

5.5. Bacteriological Tests

The diagnostic gold standards are certainly the MTB culture and the polymerase
chain reaction (PCR) but the difficulty, for several reasons, in obtaining appropriate ex-
amination samples in children represents the major obstacle to this investigation, since
the disease is often paucibacillary and children, unlike adults, hardly produce sputum
and the bronchoscopy has considerable limitations [94–96]. Therefore, non- or minimally
invasive tests on sputum (the emission is favored by performing an aerosol with hypertonic
Microorganisms 2023, 11, 1722 9 of 16

salic solution), on gastric aspirate, nasopharyngeal secretion, on urine and recent feces, are
recommended [97]. Recent studies have shown that the examination of multiple samples
increases the diagnostic sensitivity [98]. In children, due to low bacterial load, the MTB
culture has a sensitivity only of 7–40% and requires at least 2–4 weeks for growth, which
limits the use of this assay [27]. It is strongly recommended to carry out the culture by
combining solid and liquid media [99].

5.6. Radiological Investigations

The first line investigations are radiography and ultrasound of the affected site but
both may have limitations and low sensitivity, especially ultrasound since it is a highly
operator-dependent imaging modality [100,101]. Especially in the first years of life, the
radiological picture can be confused with other diseases and the thymus silhouette can
complicate the evaluation of the mediastinum when enlarged lymph nodes are present. It
is always advisable to perform an anteroposterior and lateral chest X-ray [102]. A Com-
puted Tomography scan and Magnetic Resonance Imaging may often be needed [103,104].
Radiographic changes suggestive of TB include [105]: enlargement of perihilar or paratra-
cheal lymph nodes, alveolar thickening, pulmonary miliary, cavitations (more frequent in
adolescents) and pleural or pericardial effusions. In the case of pulmonary tuberculosis,
chest X-rays should be repeated 1–2 months after the beginning of therapy. In some cases,
a worsening can be observed at the first X-ray control, despite the presence of an evident
improvement in the clinical picture. In these cases, it is advisable to repeat the X-ray after
1–2 months and at the end of therapy [106].

6. Therapy
The main objectives of therapy are to prevent the progression from infection to disease
and to cure the active form, in view of the fact that the risk of the onset of serious forms
remains high even 2 years after infection and is particularly high in children aged <2 years.
The drugs used in the treatment of susceptible and resistant TB are the same used in adults,
they vary in pediatric age both in doses and combinations. The drugs classification is
shown in Table 7.

Table 7. Drugs classification.

Group A Group B Group C

Ethambutol, Amikacin/streptomycin
Delamanid,
Levofloxacin/moxifloxacin Clofazimines Ethionamide/prothionamide
Bedaquiline Cycloserine/terizidone Pyrazinamide, P-aminosalicylic acid,
Imipenem, Meropenem,
Amoxicillyn + clavulanic acid

Compared to adults, children have fewer side effects related to therapy. Indeed, the
risk of hepatotoxicity is greater only in obese children (hepatic steatosis) and with the
concomitant administration of anticonvulsant drugs. For these children, it is advisable
to determine liver function before starting treatment and 1–2 months after the beginning.
Isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), ethambutol (EMB), rifabutin and
rifapentine are the drugs mostly used both in the therapy of latent infection and in the
active disease. Recently, it has been recommended to increase the dosage of some of these
drugs: IH 10–15 mg/Kg, RIF 10–20 mg/Kg, PZA 30–40 mg/Kg and EMB 15–25 mg/Kg [6].

6.1. Latent Infection

The latent infection is symptomatically silent and can be suspected if there has been
contact with an adult with the active disease.
Various therapeutic regimens are currently proposed [107,108]:
Microorganisms 2023, 11, 1722 10 of 16

(1) Isoniazid + rifapentine:

• Age: 2–11 years;
Isoniazid 25 mg/Kg (max 900 mg) + Rifapentine 5 mg/kg 1 dose/week per 3 months
(12 doses)
• Age ≥ 12 years;
Isoniazid 15 mg/Kg (max 900 mg): 1 dose/week per 3 months (12 doses) Rifapentine
weight Kg: 10–14: 300 mg 14.1–25: 450 mg 25.1–32: 600 mg 32.1–49.9: 750 mg ≥50:
800 mg
This regimen is not recommended in children < 2 years of age and has the same
preventative effects as isoniazid treatment for 6 or 9 months. It is preferred over isoniazid
monotherapy in children with a high risk of hepatotoxicity.
(2) Rifampicin 15–20 mg/Kg/die (max 800 mg/die) per 4 months. It has similar results
to 6 or 9 months of isoniazid [109];
(3) Isoniazid 25 mg/Kg/die + Rifampicin 15–20 mg/Kg/die per 3 months. It is rec-
ommended in children aged <5 years since the benefits of using this combination
significantly outweigh the risks;
(4) Isoniazid 10–15 mg/Kg/die (max 900 mg die) for 6 months; Isoniazid 20–30 mg/Kg/die
(max 300 mg/die) 2 times/week for 6 months;
(5) Isoniazid 10–15 mg/Kg/die (max 300 mg/die) for 9 months;
(6) Isoniazid 20–30 mg/Kg/die (max 900 mg/die) 2 times/week per 9 months.
Isoniazid prophylaxis has greater hepatotoxic effects than rifampicin and rifapentine
therapy.
During therapy, it is advisable to perform a follow-up after the first month in order to
evaluate:
• The presence of symptoms or signs indicative of progression towards the disease;
• The presence of adverse effects to the administered drugs and it is advisable to perform
a blood count and the dosage of transaminases.

6.2. Active Disease

The drugs used in the therapy of the active disease are:
• Isoniazid (H) 10 mg/Kg/die (max 300 mg/die);
• Rifampicin (R) 15 mg/Kg/die (max 600 mg/die);
• Pyrazinamid (Z) 35 mg/Kg/die;
• Ethambutol (E) 20 mg/Kg/die.
Therapy schemes [1]:
(1) Children with suspected or confirmed pulmonary tuberculosis or peripheral lym-
phadenitis living in environments with low incidence of HIV and isoniazid resistance:
HRZ for 2 months followed by HR for 4 months.
(2) Children with suspected or confirmed pulmonary tuberculosis or peripheral lym-
phadenitis or children with disseminated lung disease living in environments with
high incidence of HIV and isoniazid resistance: HRZE for 2 months followed by HR
per 4 months;
(3) Children with suspected or confirmed TB meningitis or with suspected or confirmed
osteoarticular TB: HRZE for 2 months followed by HR for 10 months;
(4) A short course of therapy (2 months with HRZ followed by 2 months with HR)
has recently been proposed in children aged 3 months to 15 years with non-severe
TB [110].
Steroid therapy (dexamethasone and prednisolone) is indicated in meningitis and TB
pericarditis. The administration of vitamin B6 is recommended both in the treatment of
latent infection and of the active disease.
Microorganisms 2023, 11, 1722 11 of 16

6.3. Treatment of Drug Resistant TB

Resistance to antituberculosis drugs is rare in children, with an estimated
25,000–32,000 children worldwide each year developing drug resistance. Several stud-
ies conducted in Europe have shown that the increase in immigration has coincided with
an increase in resistant TB cases. The diagnosis of these forms is particularly difficult in
children because it is not always possible to isolate the bacteriological material (paucibacil-
lary) to identify resistance to various drugs. According to WHO [111], four definitions of
drug resistance are distinguished: 1. Resistance to a drug, 2. Mono-resistance to isoniazid
(9%) (susceptibility to rifampicin), 3. Resistance to rifampicin (1%) [112] and, 4. Multidrug-
resistance (at least isoniazid and rifampicin) [113,114]. These children with drug resistant
bacteria are treated with the same antibiotic combinations used in adults (see Table 7).
The WHO has recently authorized the administration of two new drugs: bedaquiline and
delamanid [115]. Bedaquiline is a bactericidal agent that interferes with the ATP synthesis
of the MTB. It can be used in children of all ages. The therapy with this drug consists of
a dose of 6 mg/Kg once a day for 2 weeks followed by 3–4 mg/Kg three times a week
(max dose 200–400 mg) [116]. It is recommended for 9–12 months in multidrug-resistant
and rifampin-resistant forms. Its long half-life allows intermittent administration when
combined with other drugs used in resistant forms. Delamanid is a bactericidal agent of
the nitroimidazole class which, through nitro-reductases, reduces the synthesis of neicholic
acid and inhibits MB replication. The recommended dose is 3–4 mg/kg in one or two doses
a day in children aged <3 years, 25 mg twice a day in children aged 3–5 years, 50 mg twice
a day in children aged 6–11 years and 100 mg twice daily in ages 12–17 years [117]. It is
recommended for 6 months and can be used for a maximum of <18 months. During treat-
ment, ECG and electrolyte monitoring and neuropsychiatric monitoring are recommended
(risk of hallucinations).

7. Vaccine
Currently, BCG (Bacillus Calmette–Guerin) is still the only available vaccine in the
world [118]. It is particularly active in children in preventing the onset of the most serious
forms (meningitis and miliary) in 50% of cases and pulmonary disease in 80% of cases,
while it is unable to prevent the reactivation of the latent pulmonary infection. It can cause
disseminated forms when administered to immunodeficient and HIV positive children.
As BCG’s protection against lung infection decreases with age, there is the need for new
vaccines. Today, several new vaccines derived from killed mycobacteria, live vaccines
attenuated by genetic modifications and recombinant vaccines with viral vectors are being
tested and must complete the trial cycle to be approved [119].

8. Conclusions
TB is still a dangerous disease in childhood, as children represent the population
at greatest risk of morbidity and mortality. Furthermore, the COVID-19 pandemic has
highlighted how the decrease in access to health facilities has led to a significant delay
in diagnosis and treatment, contributing to an increase in deaths and new cases in the
following years [120]. For what concerns the diagnosis in children, it is not always simple,
especially in the first years of life, even if the introduction of new diagnostic techniques
has allowed a more rapid identification of the infection. The use of the IGRA is preferred
to the Mantoux, albeit with obvious limitations in children aged <2 years. While the
Xpert Mtb/RIF Ultra test is recommended as an initial diagnostic test of gastric aspirate
and/or stools in children with signs and symptoms of pulmonary tuberculosis. At present
it is commonly believed that there is an interrelationship between the microbiota, the
tuberculosis disease and therapy. MBT infection is linked to a peculiar and complex
immunological response, which is microbiota dependent, the profound modifications
mainly affecting the intestinal microbiota, are responsible for a lower resistance of the
subject against M. tuberculosis. From the imaging point of view, the chest X-ray is now
an important examination in children with pulmonary tuberculosis with symptoms but
Microorganisms 2023, 11, 1722 12 of 16

negative tests. Moreover, it is important to perform the lateral view in children < 5 years of
age as well.
In addition, children represent the most suitable population for brief therapy both in
the latent infection and in the active disease since a significant reduction in the side effects
is obtained.
Finally, in recent years, a greater increase in drug-resistant TB cases has been observed,
partly related to immigration from high-incidence countries. Resistance to antituberculosis
drugs is rare in children, with an estimated 25,000–32,000 children worldwide each year
developing drug resistance. It is believed that new studies are needed to better define the
therapeutic approach in drug resistant forms, especially in the age < 5 years.

Author Contributions: U.P. idealized and wrote the manuscript, R.P., S.S. and V.F., edited the
manuscript, the figures, the tables and reviewed the manuscript drafts. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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