Pediatric Tuberculosis Diagnostics: Present and Future

Abstract

The current diagnostic abilities for the detection of pediatric tuberculosis are suboptimal. Multiple factors contribute to the under-diagnosis of intrathoracic tuberculosis in children, namely the absence of pathognomonic features of the disease, low bacillary loads in respiratory specimens, challenges in sample collection, and inadequate access to diagnostic tools in high-burden settings. Nonetheless, the 2020s have witnessed encouraging progress in the area of novel diagnostics. Recent WHO-endorsed rapid molecular assays hold promise for use in service decentralization strategies, and new policy recommendations include stools as an alternative, child-friendly specimen for testing with the GeneXpert assay. The pipeline of promising assays in mid/late-stage development is expanding, and novel pediatric candidate biomarkers based on the host immune response are being identified for use in diagnostic and triage tests. For a new test to meet the pediatric target product profiles prioritized by the WHO, it is key that the peculiarities and needs of the hard-to-reach pediatric population are considered in the early planning phases of discovery, validation, and implementation studies.

INTRODUCTION

In 2020, an estimated 1.1 million children fell ill with tuberculosis (TB), but less than half were notified of national TB programs [1].

In pediatric TB, underreporting remains a substantial challenge. It not only partly reflects years of public health strategies that overlooked the importance of TB detection in children for disease control purposes but it is also a proxy for the complexities of pediatric TB diagnosis and worldwide access to appropriate diagnostics and care. The gap in pediatric TB detection is meaningful, as almost all deaths occur in undiagnosed children who are not offered anti-TB treatment [2]. The need to prioritize childhood TB detection as part of the global strategy to end TB is universally recognized, as is the urgency for research to prioritize the development of accurate and accessible diagnostic and screening tools for pediatric TB [3].

The aim of this review is to provide an up-to-date overview of the available diagnostic tools for pediatric TB and explore the panorama of future diagnostics while considering critically the challenges to implementation and priorities.

PECULIARITIES AND CHALLENGES OF DIAGNOSING PEDIATRIC TB

The difficulties of diagnosing TB in children are multifactorial. The spectrum of pediatric TB presentation is age-dependent, with the most vulnerable young children presenting often with paucibacillary intrathoracic or disseminated disease and non-specific symptoms [4]. The variety of clinical presentations and lack of pathognomonic features complicate clinical diagnosis and case definitions. Chest X-ray (CXR) is a useful diagnostic tool but is limited by inter-reader variability, and cost and accessibility issues in peripheral settings [5]. Bacteriological confirmation should be sought whenever possible [6], but the complexity of specimen collection and the low bacillary load of the samples are frequent obstacles to this approach.

Expectorated sputum is hardly collectable in young children, and obtaining “classic” pediatric respiratory specimens (gastric aspirate, GA; induced sputum, IS) requires somewhat invasive procedures, trained personnel, and equipment which are rarely available at the primary healthcare level (PHCL) [7]. Less invasive methods to collect respiratory secretions, such as nasopharyngeal aspirate (NPA), stools, and string tests are available [8] (Table 1).

Table 1.

Characteristics of Pediatric Specimens For Mycobacteriological Diagnosis.

Specimen type	Description			Xpert Diagnostic Accuracy1	Limitations	Advantages
	Principle	Procedure	Setting
Sputum (expectorated)	Collection of spontaneously produced respiratory secretions	Cooperative, older children are asked to produce sputum	Inpatient and outpatient settings	SE: 65% SP: 99% SE: 73% (Ultra) SP:97% (Ultra)	•Requires collaborative, older children and productive cough	• Cheap collection, limited supply needs • Usually available in peripheral healthcare centers
Sputum (induced)	Collection of induced respiratory secretions	Nebulization with hypertonic saline followed by nasopharyngeal suction	Inpatient and outpatient settings		• Risk of aerosolization– Requires ventilated rooms • Requires trained personnel and equipment • Not indicated in children with respiratory distress	• Criterion-standard specimen • Less invasive than gastric aspirate
Gastric aspirate	Collection of swallowed respiratory secretions	Fasting, early morning gastric content is aspirated via nasogastric tube	Inpatient (preferred) and outpatient settings	SE: 73% SP: 98% SE: 64% (Ultra) SP: 95% (Ultra)	• Invasive sample • Requires fasting • Requires trained personnel	• Criterion-standard specimen
Nasopharyngeal aspirate	Respiratory secretions collected in retropharynx	Nasopharyngeal secretions suctioned via mucus aspirator	Inpatient and outpatient settings	SE: 46% SP: 100% SE: 46% (Ultra) SP: 98% (Ultra)	• Requires trained personnel and specific equipment (suction machine and mucous trap)	• Less invasive than gastric aspirate
Stool	Collection of swallowed respiratory secretions	Spontaneously passed stools are collected	Inpatient and outpatient settings	SE: 61% SP: 98% SE: 53% (Ultra) SP: 98% (Ultra)	• Few children might pass stool during outpatient visit, requires caregivers to return to clinic • Risk of lower acceptability in older children	• Non-invasive sample • Cheap collection, limited supply needs • Does not require trained personnel • Collection might be adopted in peripheral centers
String Test	Collection of swallowed respiratory secretions	Dissolvable capsule containing a string is swallowed after 8-hours fasting. The string unravels into the stomach, where it collects swallowed respiratory secretions	Inpatient and outpatient settings	SE: 48–100%2 SP: 100%2	• Risk of low compliance in children below 4 years, unable to swallow capsule • Long procedure (up to 4-hours intragastric downtime) • Requires fasting	• Less invasive than gastric aspirate • Requires minimally trained personnel
Oral swab	Collection of material from oral cavity	Whole oral cavity is swabbed for several seconds	Inpatient and outpatient settings	SE: 30%3 SP: 100%3	• Experimental use in TB diagnostics • Suboptimal diagnostic accuracy	• Minimally invasive • Suitable as self-collection test in older children
Bioaerosols	Collection of bioaerosol in face masks or blow tubes	Face mask containing an absorptive sampling matrix (eg, gelatin or poly vinyl alcohol) is worn for 30 min Breathing for 2–5 minutes into a tube fitted a filter membrane capturing aerosols from exhaled breath	Inpatient and outpatient settings	Pediatric data not available	• Experimental use in TB diagnostic • No pediatric prototypes • Safety concerns for children below 2 years • Long collection • Risk of poor tolerance in young children	• Potential application in diagnosis and screening strategies in hard-to-reach populations • Might provide information on transmissibility of the asymptomatic

References: [6, 8, 12, 29, 44, 46].

Abbreviations: SE, sensitivity; SP, specificity.

Against a microbiological reference standard (on respiratory secretions).

Two studies [8, 29].

One study, in-house PCR [44].

Diagnostic Tools for Microbiological Diagnosis

Microscopy and Culture

Smear microscopy of sputum samples, the cornerstone of adult TB diagnosis, shows a sensitivity as low as 7% in children less than 15 years old, falling below 1% in children aged 0-4 years [9]. Mycobacterial culture is considered the gold standard of laboratory TB diagnosis, providing definitive evidence of viable Mycobacterium tuberculosis bacteria (Mtb), and enabling phenotypic drug sensitivity testing (DST) [10]. Automated liquid cultures have improved turnaround times and detection rates [11], but culture remains an expensive, sophisticated, and long procedure for tertiary-level laboratories. At best, only one-third of children with clinically diagnosed TB are culture-positive, but culture remains the most sensitive test [10].

Rapid Molecular Tests

The past decade has seen significant progress in molecular diagnostics, driven by the development and implementation of automated nucleic acid amplification tests (NAAT). (Table 2)

Table 2.

Novel Diagnostic Tests for Pediatric Intrathoracic TB Detection (Commercially Available Or In Mid/Late-Stage Development).

Assay	Diagnostic Platform/Tool	Technology	2021 WHO Recommended Use (Pediatric)	Recommended Specimen Types	Diagnostic Accuracy (MRS)	Turnaround Time	Target Setting	Target/Intended Use (Pediatric)	Commercial Availability
Xpert MTB/RIF	GeneXpert (Cepheid, USA)	qPCR	Initial test in children with signs and symptoms of TB (strong recommendation)	Sputum, GA, NPA, stool	SE: 46–73% SP: 98–100% SE: 90% (RR) SP: 98% (RR)	<2 h	District or subdistrict laboratory	Diagnostic	Yes
Xpert MTB/RIF Ultra	GeneXpert (Cepheid, USA)	qPCR/melting temperature analysis	Initial test in children with signs and symptoms of TB (strong recommendation)	Sputum, GA, NPA, stool	SE: 46–73% SP: 95–100%	<2 h	District or subdistrict laboratory	Diagnostic	Yes
Truenat MTB, MTB plus, MTB-RIF Dx. Under development: MTB-Ultima, MTB-INH, MTB-BDQ, MTB TB-COVID-19	Truenat (Molbio)	Micro RT-PCR	Initial test in children with signs and symptoms of TB (conditional recommendation)	Sputum	No pediatric data	<1 h	Peripheral laboratory (potential point-of-care)	Diagnostic	Yes
Loopamp MTBC assay	LAMP assay kit (Eiken Corp.)	Loop-mediated isothermal amplification	Currently not recommended in children	Sputum	SE: 80%1 SP: 100%1	<2 h	Peripheral laboratory	Diagnostic	Yes
LAM Ag assay	Determine TB LAMAg test (Abbott)	Lateral flow	Initial test to assist diagnosis in: – all HIV-positive children with advanced disease, regardless of TB-suggestive symptoms; - all HIV-positive children with TB-suggestive symptoms or seriously ill	Urine	SE: 43–65% SP: 57–91% (HIV-positive children)	<1 h	Point-of-care (Inpatient and outpatient)	Diagnostic (rule-in in high-risk group)	Yes
Moderate complexity automated NAAT	Several platforms (Roche, Abbott, Hain, BD, Bioneer)	High throughput molecular PCR/assay	Initial test in children with signs and symptoms of TB (conditional recommendation)	Respiratory samples	No pediatric data	6–8 h	Reference or district laboratory	Diagnostic	Yes
NGS	Several platforms (Nanopore, Illumina, ThermoFisher) and assays [Deeplex Myc-TB (GenoScreen); DeepChek (ABL)]	Targeted NGS/deep sequencing assays/WGS	NA	Sputum, culture isolates	NA	<48 h	Reference or district laboratory	Genotypic drug susceptibility testing	Yes
Second-generation LAM Ag assay	SILVAMP TB LAM (Fujifilm, Japan)	Lateral flow	NA	Urine	SE: 42–65% SP: 84–92%	<1 h	Point-of-care	Diagnostic (rule-in in high-risk group)	No
TB detection assays for ultrarapid portable NAAT	LumiraDx (LumiraDx)	Rapid molecular qSTAR/assay	NA	First product will use tongue swabs	NA	<30 min	Point-of-care	Diagnostic	No (LumiraDx platform: yes)
RNA signature assays	Xpert-MTB-HR (Cepheid), RISK6 signature assay (QuantuMDx)	Host RNA response/PCR	NA	Blood (Fingerprick)	No pediatric data	<1 h	Point-of-care (subject to instrument availability)	Triage	No
TAM TB assay	TAM-TB (Beckman Coulter)	Flow cytometry/TB-specific biomarkers (CD38 and CD27)	NA	Blood	SE: 83%1 SP: 97%1	24 h	District laboratory	Diagnostic	No

References: [6, 12, 18–20, 33–35, 55].

Abbreviations: GA, gastric aspirate; INH, isoniazid; MRS, microbiological reference standard; NGS, next-generation sequencing; NPA, nasopharyngeal aspirate; RIF, rifampicin; RR, rifampicin resistance; SE, sensitivity; SP, specificity; WGS, whole genome sequencing.

One study.

Xpert MTB/RIF (Cepheid, USA) is a low-complexity, cartridge-based NAAT running on the GeneXpert platform (Cepheid, USA), recommended for the initial diagnosis of pediatric TB and rifampicin-resistance since 2013. As with smear microscopy and culture, the test sensitivity in children is lower than in adults and varies with specimen type (Table 1) [12]. In the initial phases of the global roll-out of Xpert, high-burden countries have faced implementational challenges [13]. Data from 21 National TB programs have not only shown improvement in the uptake of Xpert MTB/RIF but have also revealed that the technology has been under-utilized for TB detection [14]. Despite the diagnostic advantages over smear microscopy, Xpert as a standalone test has not proven able to overcome the intrinsic challenges of pediatric TB detection [7, 15]. Loop-mediated isothermal amplification (LAMP) is a higher-performance alternative to sputum smear microscopy, endorsed in 2016 by the WHO for adults [16]. Despite a fairly simple procedure, the uptake in TB-endemic countries is low [17]. LAMP cannot detect drug resistance and to date the WHO does not yet recommend it in children [6]. Recent evidence suggests that the diagnostic performance in pediatric samples is comparable to Xpert [18].

Line probe assays are high complexity hybridization-based NAATs for genotypic DST, recommended for use on smear or culture-positive pediatric samples [6].

Urine Tests

Lipoarabinomannan (LAM) is a lipopolysaccharide of the mycobacterial cell wall, released from Mtb and excreted in urine. The Alere Determine TB LAM (AlereLAM; Abbott, USA) is a rapid point-of-care lateral flow assay recommended in children living with human immunodeficiency virus (CLHIV) since 2016 [6] (Table 2).

AlereLAM showed a suboptimal diagnostic accuracy in CLHIV and adults. Nonetheless, it was recommended by the WHO, considering that a large-scale roll-out would have a significant impact on mortality reduction through early TB detection and treatment of high-risk groups [19, 20].

TOOLS FOR DIAGNOSING PEDIATRIC TB IN THE 2020S: WHAT’S NEW?

New WHO-Endorsed Rapid Microbiological Tests

Initial Diagnostic Tests

Xpert MTB/RIF Ultra (Ultra; Cepheid, USA).

Ultra is the new-generation Xpert MTB/RIF assay. Ultra runs on the same GeneXpert platform, at the same unit cost (FIND-Cepheid negotiated prices for the public sector in eligible countries) [21]. The assay has a new result category, “trace call,” indicating the presence of Mtb at the lowest limit of detection, which should be interpreted as a positive finding in children [6]. “Trace” detection might increase sensitivity in pediatric TB. Ultra-demonstrated superior sensitivity compared with Xpert MTF/RIF in pediatric sputum samples (pooled sensitivity 73% versus 65%) and has been endorsed by WHO for testing of GA, NPA, IS, and stool (Table 1) [6, 12]. Regarding the assay’s diagnostic accuracy, WHO currently recommends Ultra with moderate (GA and stool), low (IS), and very low (NPA) certainty of evidence [6]. Further studies evaluating the diagnostic performance of Ultra in pediatric specimens are ongoing.

Truenat™.

Truenat MTB, MTBplus, and MTB-RIF (Molbio, India) are chip-based micro-PCR assays that function on a compact, battery-operated device that requires minimal user input [6] (Table 2). Truenat showed comparable diagnostic accuracy to Xpert on sputum samples [22], with the advantages of truly portable hardware. Following a positive Truenat MTB or MTBplus test, MTB-RIF can be used as a reflex test to detect rifampicin resistance. All types have been conditionally endorsed by WHO for use in pediatric sputum specimens [6]. To date, there are, however, no published studies assessing test performance in children or on samples other than sputum.

Other Initial Diagnostic Tests.

Moderate-complexity automated NAAT are high-throughput assays that run on established multi-pathogen platforms and can simultaneously detect isoniazid and rifampicin resistance. The indications for use in children are extrapolated from adult data, anticipating that diagnostic performance might be lower in paucibacillary cases [6] (Table 2).

Follow-on Diagnostics for Additional Drug-Resistance Detection

This category includes the new Xpert MTB/XDR for detection of resistance to isoniazid and second-line anti-TB drugs, and high complexity hybridization-based NAAT for the detection of pyrazinamide resistance on culture isolates. Both assays are conditionally recommended in children [6].

Novel Specimens and Combination of Specimens for Microbiological Diagnosis

In 2021, testing of stool specimens for the initial diagnosis of childhood TB with Xpert/Ultra was recommended by the WHO, based on recent meta-analyses [23]. However, stool sensitivity is heterogeneous and might be lower in younger children [24]. In addition, stool specimens need processing before testing; but now several methods exist, including a simple one release-and-sedimentation step entirely using the Xpert/Ultra equipment [25]. The techniques showed comparable performances in experimental settings [26], and a diagnostic trial is ongoing [27]. Repeated sampling might increase diagnostic yield by up to 33% [28]. In addition, the collection of minimally invasive specimens to increase Xpert sensitivity is a promising strategy. One recent study showed that the combination of two NPAs (sensitivity 74%), or one NPA, and one stool sample (sensitivity 71%), performed comparably to criterion-standard specimens (two IS: 64% sensitivity; two GA: 77% sensitivity) in children younger than five years [29].

Novel Clinical Algorithms

Laboratory-based diagnosis in resource-limited settings remains challenging, and it is vital to identify approaches to refine the clinical decision-making processes to consider and diagnose TB at the PHCL more accurately. In HIV-uninfected children, a treatment-decision algorithm based on clinical assessment identified 71% of children with confirmed TB. The clinical evaluation included the history of TB exposure and symptoms (persistent cough, fever, weight loss/failure to thrive, lethargy, and hepatomegaly). The algorithm showed a sensitivity of >90% across all age groups, and a specificity of 52% if including CXR and Xpert MTB/RIF in the model [30]. A similar performance was seen in a treatment-decision score based on clinical and radiological findings in CLHIV (sensitivity: 89%; specificity: 61%) [31]. While clinical algorithms might represent valuable rule-out approaches, the role of radiology and Xpert to achieve greater specificity confirms the limitations of symptom-based diagnosis and the need for integrated strategies to minimize misdiagnosis.

WHAT IS IN THE FUTURE OF PEDIATRIC TB DIAGNOSTICS?

Recent advancements provide a reason for optimism, but the current diagnostic tools are still suboptimal or rarely available at the PHCL. The target of closing the pediatric TB detection gap is unlikely to be met without novel, more accurate diagnostics that can be used in affordable diagnostic algorithms, ideally at point-of-care or close to point-of-care [3, 32]. The specific challenges of pediatric TB diagnosis should ideally be considered in early-phase designs for a product (or strategy) to successfully meet the minimal diagnostic accuracy thresholds of target pediatric product profiles (TPP) set by the WHO to positively impact TB detection and outcomes [32]. Hence, the development of more sensitive pathogen-based assays for use on non-respiratory specimens, and the expansion of tests that use pediatric-specific biomarkers of the host response to Mtb are considered high priorities. Several novel assays and technologies at various stages of development hold promise using these approaches.

Pathogen-Based Assays

Assays for the Detection of Mycobacterial Antigens

FujiLAM and Ultra-Sensitive LAM.

The SILVAMP TB LAM test (FujiLAM, Fujifilm, Japan) is a second-generation lateral flow assay that uses high-affinity monoclonal antibodies and a silver-amplification step [33]. Pediatric studies have shown varying accuracy (Table 2), with a high specificity observed in malnourished children and CLHIV, for which FujiLAM might have a useful role as a rule-in test for children with a high pretest probability of disease [33, 34]. Ultra-sensitive LAM tests, using improved reagents and novel assay designs, are currently in early developmental stages [35].

Assays for the Detection of CFP-10 and ESAT-6.

In a proof-of-concept study, a high-throughput mass spectrometry assay for the detection of CFP-10 and ESAT-6 derived antigens in blood demonstrated high sensitivity and specificity in culture-negative (clinically diagnosed) adult TB patients [36]; the performance of pediatric specimens is currently being studied, but if adult results were to be confirmed, detection of Mtb circulating peptides could represent a promising non-sputum-based diagnostic approach for patients with the paucibacillary disease.

Molecular Diagnostic Assays

Point-of-Care NAAT.

Decentralization of pediatric TB services is a WHO priority [3]. Decentralizing molecular testing proved effective for same-day test-and-treat and improved disease outcomes in adults [37]. Battery-powered, ultrarapid point-of-care NAAT are in various stages of development and may represent useful tools for test decentralization strategies, ideally in combination with minimally-invasive, non-sputum specimens [35]. Operational research and modelling exercises will be key to understanding feasibility and impact on pediatric populations.

Next-Generation Sequencing (NGS).

NGS-based assays can provide information on the whole mycobacterial genome (WGS) or identify gene regions of interest with targeted NGS. Sequencing can detect mixed infections or re-infections with different strains, and inform about transmission chains [38]. NGS is emerging as a tool for DST, as compared with culture and probe-based assays. NGS and WGS are powerful tools for quick, accurate and extensive genome profiling and can also distinguish silent mutations [39]. End-to-end solutions using targeted sequencing assays that do not require DNA extraction from culture isolates are expanding [40]. NGS technology is limited by high-costs, the need for expertise and robust bioinformatic systems. Capacity has been greatly improved through the COVID-19 pandemic and portable sequencing devices are now available. Portable NGS tools will become increasingly useful in the future, but the road to large-scale applicability for DST in endemic countries is still long [41].

Circulating Free DNA (cfDNA).

cfDNA is gaining momentum as a microbial biomarker in infectious disease diagnostics [42]. In a proof-of-concept study, Mtb cfDNA was found in the plasma of non-mycobacteremic patients with smear-positive TB. Pending validation in pediatric cohorts, cfDNA assays might become an alternative to point-of-care diagnostics on respiratory specimens [42].

Novel Specimens for Molecular Diagnostic Assays

The WHO policy inclusion of stool samples for testing with Xpert and the high sensitivity achieved by combinations of minimally invasive specimens mark the importance of expanding research on child-friendly samples. Mtb is detectable in adult patients with TB using oral swabs (OS) with fair accuracy [43], however, in one pediatric study, the sensitivity of one OS with an in-house PCR was low (30% against the microbiological reference standard) [44]. Another recent study showed that Ultra on pediatric OS had a low sensitivity (22%) against microbiologically confirmed TB on IS [45]. Bioaerosols sampling, for example, in face masks (FM) has been suggested for the detection of exhaled Mtb and other respiratory pathogens [46]. This simple method can be used in combination with existing NAATs and it showed high sensitivity to detecting subclinical disease in an active case-finding pilot study [46]. There are currently no studies assessing the feasibility of FM sampling in children, however, FM and OS appear to be child-friendly collection methods that have the potential to be implemented at the PHCL on point-of-care NAAT (Table 1).

Assays for the Detection of Breath Metabolites

Several studies have demonstrated the biomarker potential of volatile organic molecules (VOC) in exhaled breath of individuals with TB. In a recent study, a 4-compound VOC signature distinguished children with TB with high accuracy [47]. African giant pouched rats trained to “sniff” TB on pediatric sputum samples significantly increased case detection [48], but the feasibility and end-user acceptability should be determined in large-scale programmatic interventions before considering the use of trained rats as a first-line TB screening approach. “E-nose” devices are also undergoing evaluation and some prototypes have shown promise as screening tests in adults, but pediatric data are still lacking [49].

Host-Based Assays

Assays Based on Host RNA Response

Host RNA expression patterns in response to Mtb can be used to identifying patients with TB; in a cohort of African children a 51-transcript signature distinguished TB from other diseases with a sensitivity just below the TPP threshold for a triage test, and with good specificity [50]. Data from this study and adult datasets contributed to identify a 3-transcript signature [51]. A point-of-care, cartridge-based prototype leveraging the GeneXpert platform, measure this signature in the capillary, for example, finger-prick, whole blood to generate a “TB score” to distinguish active TB [52]. In an interim analysis, the assay achieved the minimal TPP for a triage test in adults [52]. Pediatric studies on this host-response cartridge are ongoing. Other RNA signatures have been found to have high diagnostic accuracy in children and warrant further validation studies [53]. In adults, transcriptional signatures are also promising correlates of the risk of disease progression [54]. Transcriptomic biomarker-based point-of-care assays for incipient TB and treatment monitoring are also in development [35].

T-cell activation marker TB assay (TAM-TB) Assay

The T-cell activation marker TB assay (TAM-TB) is a flow-cytometry assay that measures the differential expression of activation and maturation markers on CD4+T-cells in response to Mtb infection [55]. In a proof-of-concept study among children in Tanzania, it met the TPP for a diagnostic test [55]. Commercial TAM-TB assays are in development and pediatric studies are ongoing [35]. Large prospective studies to evaluate TAM-TB performance in the field are underway, but to date it remains an expensive and rather complex technique, not suitable for the PHCL. However, one recent study demonstrated the successful implementation of the assay at the district level in a resource-limited setting [56], and it is desirable that flow-cytometry-based assays could be made more portable, similar to the instruments for rapid molecular diagnosis.

Chemokine and Cytokine Biomarkers for the Diagnosis of TB

Research activities focusing on identifying candidate cytokine and chemokine biomarkers from antigen-stimulated (to elicit Mtb-specific responses) or unstimulated blood samples as correlates to improve pediatric TB diagnosis are an active field. In one study, IL-2, IL-13, and CXCL10 (also known as IP-10) emerged as high-performance cytokines to discriminate TB from other diseases in TB-exposed children; IL-1ra and TNF-α levels differed significantly between active and latent TB, suggesting a potential role as stage-specific biomarkers [57]. In another study, IL-1ra, IL-7, and IP-10 were distinguished with good accuracy children diagnosed with TB from other respiratory illnesses, and might constitute a candidate for use as a triage test in children [58].

Exosomal Markers for the Diagnosis of TB

Exosomes are extracellular vesicles released by cells in both physiological and stress conditions such as infections. Exosome-associated molecules are increasingly studied as potential diagnostic TB biomarkers. Proteins and microRNA associated with exosomes derived from Mtb-infected cells have been identified in various biospecimens, including in one pediatric study, and omics approaches might help to further identify the most promising candidate biomarker signatures [59].

Novel Specimens for Host-Based Assays

Saliva is rich in immune response proteins [60]. Adult studies suggest that salivary cytokine and other protein biosignatures are promising TB screening biomarkers, but pediatric-specific ones have yet to be identified. Saliva collection is child-friendly, and this specimen would be well suited for a potential host-based triage test [60].

The Role of Omics in Diagnostics Discovery

As outlined above, promising and significant progress has been made in the field of transcriptomics for TB diagnostics in the last decade [50, 51, 53]. In recent years, other omics fields including proteomics, metabolomics, and lipidomics have gained increasing interest. Deep plasma profiling is possible thanks to novel techniques of mass spectrometry, functional proteomics, and robust bioinformatics. One recent study provided a comprehensive profile of a TB plasma proteome, identifying a promising 5-protein panel for diagnosis of active TB among adults [61]. In addition, proteomic signatures have shown promise as potential tests to identify individuals at increased risk of progression to active TB [62]. The discovery of novel analytes is not limited to the immunoproteome: proteomic microarray approaches are increasingly used to identify Mtb peptides that might discriminate between latent and active infection [63]. Pediatric proteomic data are limited, but studies are currently underway.

The field of metabolomics studies metabolic dysregulations in the host associated with TB. Recent pediatric studies have identified panels of blood metabolites that distinguished TB from other diseases [64, 65], and showed good diagnostic accuracy for TB [66], suggesting potential future applicability of host metabolites in triage tests for children.

As demonstrated with LAM, species- and strain-specific lipoglycans and mycolic acids might represent suitable diagnostic biomarkers. The advances in mass spectrometry techniques have expanded to lipidomics, leading to the creation of libraries of newly identified mycobacterial lipids that need further characterization but hold promise for use in diagnostics [67]. Host lipidomic profiling is also emerging as the role of pro-inflammatory lipidic mediators in TB susceptibility and immunopathogenesis is being delineated [68]. Pediatric studies in this area would be desirable.

However, high costs and complexity limit the use of omics technologies. Pediatric datasets have been rarely included in discovery studies so far, and further clinical studies in different pediatric populations are needed to validate adult signatures and derive pediatric-specific ones. Nonetheless, omics approaches are a valuable tool to improve our multidimensional understanding of complex host-pathogen interactions and are emerging as potentially key tools specifically for novel biomarker discovery and the development of novel TB diagnostics.

Digital Health

Computer-Aided Detection

Computer-aided detection (CAD) proved to be an accurate tool that can replace human reader interpretation of CXRs among adults and also overcome inter-reader variability [69]. Although some manufacturers have claims for use in children above the age of five years [65], there are currently no data on the application of CAD to pediatric radiographs and the peculiarities of disease presentation warrant an evaluation on pediatric CXR libraries. CAD also has the potential to be expanded to ultraportable digital X-ray systems, thus potentially becoming an important tool for active case finding and testing decentralization strategies in low-resource settings [70].

AI-Enabled Point-of-Care Lung Ultrasound

Point-of-care lung ultrasound is recognized as a potentially valuable diagnostic approach and holds promise for the detection of pulmonary and extrapulmonary TB in children [71]. Preliminary studies on children with pneumonia suggest that AI-enabled point-of-care lung ultrasound may become an option in the future [72].

CONCLUSIONS

Although pediatric TB still represents a diagnostic challenge, the 2020s have witnessed unprecedented progress. Portable point-of-care NAATs for service decentralization, novel non-respiratory specimens, and combinations of child-friendly samples warrant further exploration, likely in integrated strategies, to identify the most effective and feasible diagnostic and screening approaches. Clinical algorithms are sensitive screening tools for PHCL, whose specificity could be improved by embedding more sensitive and true point-of-care rapid microbiological or host-based biomarker tests or CAD. Nevertheless, many assays in the most advanced stages of development urgently need validation in pediatric populations and are far from the wide-scale application. In the search for the next-generation of pediatric diagnostic testing approaches, it is key that children are included in early-phase studies and that operational challenges are promptly addressed. The ideal test must demonstrate a clear diagnostic and therapeutic impact, and ultimately improve patient outcomes. Pediatric TB is treatable, and timely and accurate detection can increase access to shorter and child-friendly regimens [23], avoid evitable deaths, and contribute to TB control globally.

Notes

Financial support. F. W. B. and R. S. were supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers NIH/NIAID 1UO1AI52084-01 and NIH/NIAID 1RO1AI152159-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Supplement sponsorship. This article appears as part of the supplement “What’s New in Childhood Tuberculosis?” sponsored by the Stop TB Partnership.

Potential conflicts of interest. P. N. and M. R. are employed by FIND, Geneva, Switzerland, a not-for-profit foundation that supports the evaluation of publicly prioritized tuberculosis assays and the implementation of WHO-approved (guidance and prequalification) assays using donor grants. FIND has product evaluation agreements with several private sector companies that design diagnostics for tuberculosis and other diseases. These agreements strictly define FIND's independence and neutrality with regards to these private sector companies. F. W. B. and R. S. have nothing to disclose.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Author notes

These authors contributed equally.