Key messages
- Using both respiratory samples and stool for low-complexity automated testing (LC-aNAATs) increased the number of pulmonary tuberculosis cases detected in children (with and without HIV).
- Adding the lateral flow urine lipoarabinomannan test (LF-LAM) identified additional pulmonary tuberculosis cases in children with HIV.
- Parallel testing may lead to more misdiagnoses of children who do not have tuberculosis (false positives), and this undesirable effect is likely more pronounced in settings where tuberculosis is less common.
Why is it difficult to diagnose pulmonary tuberculosis in children?
Children with tuberculosis of the lung (pulmonary tuberculosis) have non-specific symptoms that make it hard to diagnose. The standard approach to diagnosis of pulmonary tuberculosis is to detect the bacteria (Mycobacterium tuberculosis) in the lungs by collecting sputum. However, children often cannot cough up sputum and require more complicated procedures to get a respiratory sample. In addition, children have a low number of bacteria in their sputum compared to adults, making the tests less useful than they are for adults. This is particularly true for children with HIV.
What is parallel testing?
Parallel testing refers to using more than one test at the same time. This could mean using two or more tests, or two or more types of samples with the same test. A positive parallel testing result means any one of the tests returned a positive result. We studied parallel testing using both respiratory and stool samples with LC-aNAATs to detect the tuberculosis bacteria. For children with HIV, we also studied the effect of adding a urine test for tuberculosis (LF-LAM).
What did we want to find out?
We wanted to find out:
- the accuracy of parallel testing for pulmonary tuberculosis in children; and
- whether parallel testing increased the accuracy of detecting childhood pulmonary tuberculosis compared to a single test.
What did we do?
We searched for studies that carried out these multiple tests in children who were being tested for pulmonary tuberculosis. We combined the results across these studies, and we looked at the accuracy of these tests alone and when used in parallel. We then determined if parallel testing was able to identify more cases of tuberculosis in children than one test alone. We also determined whether parallel testing led to more children without tuberculosis being misdiagnosed as having tuberculosis.
What did we find?
Overall, we looked at results from 14 studies, six of which involved children with HIV. The children in the studies were from 14 different countries where tuberculosis is common.
Main results
Children without HIV or of unknown HIV status
In a theoretical population of 1000 children without HIV, where 100 children have pulmonary tuberculosis, the parallel use of LC-aNAATs on respiratory and stool samples would diagnose 7 additional children with tuberculosis, compared to if we only used LC-aNAAT on a respiratory sample. However, it will also result in 16 more children without tuberculosis being misdiagnosed as having tuberculosis.
Compared to using only LC-aNAAT on stool alone, parallel testing of LC-aNAAT on respiratory and stool samples will identify 22 more children with tuberculosis, but will misdiagnose 37 more children without tuberculosis.
Children with HIV
In a theoretical population of 1000 children with HIV, where 400 children have pulmonary tuberculosis, the parallel use of LC-aNAATs on respiratory and stool samples would diagnose 16 more children, compared to if we used only an LC-aNAAT on a respiratory sample, but would misdiagnose 11 more children without tuberculosis.
Compared to using only LC-aNAAT on stool alone, parallel testing of LC-aNAAT on respiratory and stool samples would identify 34 more children with tuberculosis, but would misdiagnose nine more children without tuberculosis.
If we added LF-LAM testing to the parallel use of LC-aNAAT on respiratory and stool samples, we would correctly diagnose 28 more children with tuberculosis than if we only performed LC-aNAAT on respiratory and stool samples, but additionally, we would misdiagnose 61 children without tuberculosis.
Tuberculosis prevalence (occurence in the population)
For children with and without HIV, if fewer children had tuberculosis in the population, then parallel testing would diagnose fewer children with tuberculosis, while misdiagnosing more children without tuberculosis.
What are the limitations of the evidence?
Although we were able to include a large number of children from different settings, we were not able to include data for some existing studies. It is challenging to diagnose pulmonary tuberculosis in children, even with the benchmark test, and this reduced our confidence that parallel testing misdiagnosed children with tuberculosis. Because of these limitations, our confidence in the evidence is generally low to moderate.
How up to date is this evidence?
The evidence is based on searches run up to 3 November 2023.
Using LC-aNAAT with both respiratory and stool samples may increase the sensitivity of diagnostic testing for tuberculosis in children, including those with HIV, and the addition of LF-LAM for children with HIV may further increase sensitivity, although at the cost of reduced specificity. Stool and urine testing is non-invasive and may complement testing respiratory samples to increase tuberculosis case detection in children. The benefits of parallel testing may be greater in settings with high tuberculosis prevalence, while there may be a larger proportion of false-positive results and greater risk of overtreatment in areas of low tuberculosis prevalence.
Low-complexity automated nucleic acid amplification tests (LC-aNAATs) are molecular assays widely used to diagnose tuberculosis disease in children. The lateral flow urine lipoarabinomannan assay (LF-LAM) is recommended for use amongst children with HIV. Previous systematic reviews have assessed the diagnostic accuracy of LC-aNAATs and LF-LAM separately in children, but in clinical practice the tests may be used concurrently, i.e. in 'parallel'.
To compare the diagnostic accuracy of the parallel use of LC-aNAAT on respiratory and stool specimens in children, and with LF-LAM on urine amongst children with HIV, versus each assay alone for detecting pulmonary tuberculosis disease.
We searched MEDLINE, Embase, Science Citation Index-Expanded, Conference Proceedings Citation Index – Science, Biosis Previews, the Cochrane Central Register of Controlled Trials, Scopus, WHO (World Health Organization) Global Index Medicus, ClinicalTrials.gov, and the WHO International Clinical Trials Registry up to 3 November 2023. There was a WHO public call for data on the accuracy of LC-aNAAT and LF-LAM for children until December 2023.
We included studies that enroled children under 10 years of age with presumptive pulmonary tuberculosis, and provided data to assess the accuracy of parallel testing and at least one of the component tests, against a microbiological reference standard (MRS) based on culture or composite reference standard (CRS) that included clinical diagnosis.
We extracted data using a standardised form and assessed study quality using QUADAS-2 and QUADAS-C tools. We performed bivariate random-effects meta-analysis using a Bayesian approach to estimate sensitivity and specificity and absolute differences between index tests. Diagnostic accuracy estimates were calculated primarily against the MRS and secondarily against the CRS. We used GRADE to assess the certainty of the evidence on comparative accuracy.
We included 14 studies to assess parallel testing in children with and without HIV. In addition, six of the 14 studies were included to evaluate LC-aNAATs with LF-LAM amongst children with HIV. Other than a high risk of bias with the CRS due to the potential incorporation of index results in clinical diagnoses, studies generally had low risk of bias across QUADAS-2 and QUADAS-C domains.
Parallel use of respiratory and stool LC-aNAATs
Children without HIV or HIV status unknown
We included eight studies (2145 participants, tuberculosis prevalence 8.1% (173/2145)) for assessment against the MRS. Parallel use of LC-aNAAT on respiratory samples and stool had an estimated pooled sensitivity of 79.9% (95% credible interval (CrI) 67.9 to 89.8) and an estimated pooled specificity of 93.4% (95% CrI 87.2 to 97.0). Compared to LC-aNAAT on respiratory samples alone, parallel testing had 7.1 (95% CrI 3.2 to 13.4) percentage points higher sensitivity and −1.7 (95% CrI −3.8 to −0.6) percentage point change in specificity (both low-certainty evidence). Compared to LC-aNAAT on stool alone, parallel testing had 22.1 (95% CrI 13.7 to 32.7) percentage points higher sensitivity (moderate-certainty evidence) and a −4.1 (95% CrI −8.0 to −1.7) percentage point difference in specificity (low-certainty evidence).
Children with HIV
Against the MRS (seven studies, 697 participants, tuberculosis prevalence 6.3% (44/697)), parallel use of LC-aNAAT on respiratory samples and stool had an estimated pooled sensitivity of 70.2% (95% CrI 51.1 to 84.7) and specificity of 95.4% (95% CrI 91.7 to 97.8). Compared to LC-aNAAT on respiratory samples alone, parallel testing had 4.0 (95% CrI 0.6 to 12.9) percentage points higher sensitivity (moderate-certainty evidence) and −1.9 (95% CrI −3.9 to −0.7) percentage point difference in specificity (moderate-certainty evidence). Compared to LC-aNAAT on stool alone, parallel testing had 8.5 (95% CrI 2.4 to 20.9) percentage points higher sensitivity and −1.4 (95% CrI −3.3 to −0.4) percentage point difference in specificity (both moderate-certainty evidence).
Composite reference standard
The parallel use of respiratory and stool LC-aNAATs had lower sensitivity than the CRS in children with and without HIV, with smaller differences compared to using each component test alone (very low-certainty evidence for children without HIV; low-certainty evidence for children with HIV). The specificity of parallel testing was similar between MRS and CRS.
Parallel use of respiratory and stool LC-aNAATs and LF-LAM amongst children with HIV
We included six studies for the evaluation of diagnostic accuracy against the MRS (653 participants, tuberculosis prevalence 6.6% (43/653)). Parallel use of LC-aNAAT on respiratory and stool samples and LF-LAM had an estimated pooled sensitivity of 77.6% (95% CrI 60.0 to 89.6) and an estimated pooled specificity of 83.9% (95% CrI 73.9 to 90.4). Compared to LC-aNAAT on respiratory and stool samples, parallel testing had 6.9 (95% CrI 1.5 to 20.1) percentage points higher sensitivity (low-certainty evidence) and a −10.2 (95% CrI −19.6 to −4.9) percentage point difference in specificity (moderate-certainty evidence).
Composite reference standard
Against the CRS (six studies, 674 participants, tuberculosis prevalence 42.4% (286/674)), parallel use of LC-aNAAT on respiratory and stool samples and LF-LAM had a pooled sensitivity of 30.0% (95% CrI 13.2 to 54.8) and specificity of 83.3% (95% CrI 69.8 to 90.2). Compared to LC-aNAAT on respiratory and stool samples, parallel testing had 11.5 (95% CrI 3.8 to 26.7) percentage points higher sensitivity (very low-certainty evidence) and −10.1 (95% CrI −21.6 to −4.9) percentage point difference in specificity (low-certainty evidence).
Liverpool School of Tropical Medicine, Foreign, Commonwealth and Development Office (FCDO)
WHO, TB Prevention, Diagnosis, Treatment, Care & Innovation (PCI), Global TB Programme
Protocol available via https://doi.org/10.1002/14651858.CD016071, version published 13 May 2024