What’s New in the Molecular Diagnosis of Childhood Tuberculosis?

Sample Collection Methods in Children and Their Diagnostic Accuracy for Tuberculosis Detection Sample collection in children to detect tuberculosis (TB) is a vital consideration in achieving a microbiologic diagnosis.1 In children who have difficulty expectorating sputum, particularly those under the age of 5 years, sample collection includes gastric aspirate/gastric lavage, which has a diagnostic sensitivity of 65%, and induced sputum, where 3 cumulative induced sputum samples can have a diagnostic sensitivity of around 85%–90%.2 Noninvasive specimens such as nasopharyngeal aspirates (NPA), stool and oral swabs are promising alternatives.3–5 In a comparison of NPAs and gastric aspirate, the diagnostic sensitivity of NPA was 50% in culture-positive patients and increased to 65% with 2 NPA samples.3 Recently, the World Health Organization (WHO) recommended Xpert Ultra (Cepheid) testing of stool in suspected pediatric pulmonary TB. In comparison to a reference standard, the sensitivity and specificity of stool Xpert Ultra testing in children with presumptive TB are 67% and 99%, respectively.4 The sensitivity is higher (79%) in children with HIV infection and smear-positive sputum but lower in younger children, potentially limiting the use of the stool as a diagnostic specimen in this age group.4,6 Oral swabs taken from the buccal or tongue mucosa are being assessed for TB detection.7 However, in children, optimization of commercial nucleic acid amplification tests (NAATs) is needed as the sensitivity of oral swabs is only 22% with Xpert Ultra.8 Other sampling methods include the Pediatric Entero-string test, which retrieves gastric fluid via a nylon string coiled in a swallowed capsule for TB testing.9 Bioaerosol sampling using masks has been shown to have high sensitivity in detecting exhaled Mycobacterium tuberculosis (Mtb) and other respiratory pathogens in adults.10 A study in Mumbai assessed the feasibility of using bioaerosols in conjunction with reverse transcriptase (RT)-PCR for TB detection in children. In this noninvasive and simple method termed SMaRT-PCR, children with pulmonary TB wore an N-95 mask with a gelatin membrane insert for 10 minutes. During this time, children talked or read or recited loudly, coughed intermittently and performed deep breathing. The aerosols exhaled/expelled by them were collected on the membrane. After sampling, the membrane was dissolved in RNA stabilization and isolation media, and processed for RNA and RT-PCR to detect TB-specific genes. Using sputum or gastric aspirate analyzed with GeneXpert as the reference standard, the sensitivity and specificity of mask sampling were 75% and 95%, respectively.11 In extrapulmonary TB, invasive procedures are required to obtain material for testing with Xpert assays. In peripheral lymphadenitis, the sensitivity is around 80%, and the specificity is 94%. Low sensitivities of 42%, 50% and 25 % are observed in cerebrospinal fluid samples, pleural fluid and ascitic fluids, respectively, with better corresponding specificities of 99%, 83% and 83%.12,13 NEWER MOLECULAR TESTS TB LAMP This assay is based on a loop-mediated isothermal amplification reaction where amplified target DNA is detected visually. A study in children found that TB Loop mediated Isothermal Amplification had 84% sensitivity and 96% specificity using positive mycobacterial culture as a reference standard.14 With only requiring basic equipment, this method is useful for TB detection in resource-limited settings. Nucleic Acid Amplification Tests Advances in molecular TB diagnostics over the last decade have resulted in emerging technologies for TB testing that are both accurate and fast. Molecular tests are also used to detect resistant target gene mutations. Low-complexity tests such as the Xpert Ultra assay are more sensitive than the previous-generation Xpert MTB/RIF assay. Xpert Ultra offers fully nested multi-target copies for a limit of detection equivalent of 10–100 CFU/mL and an improved design that enhances the specificity of the assay for rifampicin resistance.15,16 The Xpert Ultra assay significantly increases TB detection in smear-negative children.16 The Xpert XDR as a near point-of-care assay has been evaluated for isoniazid and second-line resistance detection, including fluoroquinolones and second-line injectables.17 With high diagnostic accuracy for isoniazid and fluoroquinolones, this 8-plex Polymerase Chain Reaction is poised to be a useful second-line test.17 Moderately Complex NAATs can yield Drug Susceptibility Test (DST) results faster than traditional phenotypic culture/DST.15 Line probe assays can detect point mutations in rifampicin, isoniazid, fluoroquinolones, and aminoglycosides. The canonical mutations are useful in deciding treatment options. WHO has endorsed high-throughput automated NAAT platforms, including Abbott Real-time MTB RIF/Isonaizid, Bruker-Hain FluoroType MTBDR, Becton Dickinson Max Multi Drug Resistant Tuberculosis assay and Roche Cobas MTB RIF/Isonaizid assay. A systematic review found that these systems had a sensitivity of 93%–96% and a specificity of 97%–98% for Mtb detection.18,19 Despite the fact that these platforms can detect down to 17 CFU/mL, currently no pediatric performance data are available.19 Next-generation Sequencing In contrast to probe-based assays, today highly complex assays such as next-generation sequencing (NGS) offer plausible solutions for detecting and characterizing drug-resistant TB with clinically relevant resistance-conferring variants or for analyzing whole genomes. Whole Genome Sequencing Whole genome sequencing (WGS) allows for the interrogation of the entire genetic repertoire of MTB, enabling the possibility of precision medicine, especially for drug-resistant cases.20 High-income, low-TB-burden settings, such as England, have transitioned from phenotypic culture to WGS for DST for first-line and some second-line drugs.21–23 WGS implementation in low-income, high-TB-burden settings, where it is most essential, is hampered by the need for specialized facilities with complex workflows, skilled personnel, and data analysis capability.24 Targeted NGS Targeted NGS (tNGS) assays offer the advantage of being applied directly to smear-positive samples, providing detailed and accurate sequence information for a greater number of loci than existing molecular tests, and can be performed using cloud-based analysis platforms.15 The Myc-TB assay (Deeplex), DeepChek-TB (ABL Diagnostics S.A.) and a new Oxford Nanopore Technologies assay employ sequencing of amplicon mixes to identify mycobacterial species and genotypes and determine known sequence variants in antimicrobial resistance.25,26 However, no pediatric data for these assays have been reported yet. Pyrosequencing Pyrosequencing is now being used to detect extensively drug-resistant (XDR) TB. In 1 study, pyrosequencing had 98.1% sensitivity in patients with TB meningitis (TBM) when using the TBM uniform case definition. In comparison with the Xpert MTB/RIF assay and TB culture using Mycobacteria Growth Indicator Tube, pyrosequencing of cerebrospinal fluid samples was found to be significantly more sensitive for diagnosing TBM. Furthermore, it aided early therapeutic decision-making by providing information on diagnosis and DST to the XDR-defining drugs.27 Molecular Tests Currently Under Evaluation Metagenomic NGS Metagenomic NGS (mNGS) is another novel diagnostic approach, which provides unbiased sequencing and identification of microbial genetic material.28 In 1 study that included pediatric samples, mNGS had a peak sensitivity of 55% in bronchoalveolar lavage samples which was higher than culture in the same samples (35.2%).29 In 2 studies, which included children with TB, mNGS was also used for the detection of TB meningitis.30,31 Large consortia, such as TB-SPEED (https://www.tb-speed.com), are currently evaluating these novel diagnostic approaches. Biomarkers Specific transcriptomic profiles associated with microbiologically confirmed or clinically diagnosed TB have been identified using gene signatures for pediatric TB diagnosis.32 In children, a 51-transcript RNA signature had a diagnostic sensitivity and specificity of 83% and 84%, respectively, for microbiologically proven TB.32 Another study showed that children with TB have different gene signatures than adults with TB.33 Implementation of diagnostic gene signatures necessitates additional validation in different populations, a reduction in transcript numbers for TB identification, and more suitable point-of-care platforms. Circulating Free DNA Another new approach to TB diagnosis is detecting Mtb circulating free DNA (cfDNA) in human plasma, pleural fluid and other body fluids using RT-PCR. A meta-analysis of data from 14 studies found Mtb cfDNA detection had a sensitivity and specificity of 68% and 98%, respectively, and typically only requires only a few hours until detection.34 Subsequently Mtb cfDNA could be detected in plasma samples of children via metagenomic sequencing, albeit only in 50% of the smear-positive pediatric patients (and in none of the smear-negative, culture-positive children).35 CONCLUSIONS In the last decade, there have been numerous advances regarding both high- and low-complexity molecular assays aiding the diagnosis of TB in children. However, in resource-poor countries especially, where TB and DR-TB are major problems, to have an actual impact, molecular diagnostic tests should be inexpensive with a quick turnaround time and as near to the patient as possible, so that appropriate treatment can be initiated without major delays.