Accuracy of Xpert Ultra in Diagnosis of Pulmonary Tuberculosis among Children in Uganda: a Substudy from the SHINE Trial

DISCUSSION

This diagnostic substudy among children clinically treated for minimal TB disease at the Ugandan site of the SHINE trial found that 50/353 (14.2%) of children had microbiologically confirmed TB when samples positive only by Ultra were treated as true positives. When samples positive only by Ultra were excluded, the prevalence of microbiologically confirmed TB was 33/353 (9.4%).

There was a surprisingly high number of samples (n = 17) positive only by Ultra, almost all of which were positive in the trace category. There are a number of possible explanations for this finding, and it is important to interpret this in the context of this specific study population of children (98% with minimal TB disease [i.e., all sputum smear negative]); the yield of all tests used would be lower than expected for children with sputum smear-positive TB. The smear-negative nature of this study population may affect some tests to a greater degree than others. Studies using spiked sputum samples have shown that around half of samples are positive by Ultra with as few as 2.5 CFU/ml (5, 6). Thus, it is possible that samples positive only by Ultra may have had a bacillary load too low to be detectable by Xpert (LOD, ∼116 CFU/ml) and potentially also too low for culture (LOD, >10 CFU/ml). Another explanation is that bacilli were rendered nonviable (and thus culture negative) by gastric acid, by delays in inoculation of the culture medium, by antituberculosis therapy initiated before the time of testing, or by host inflammatory response. In a recent study evaluating the diagnostic value of Ultra in childhood tuberculosis using high-quality respiratory samples (bronchoalveolar lavage fluid), 58% of Ultra-positive samples were negative on both smear microscopy and culture (16).

Conversely, one could view the samples that were positive only by Ultra as false positives. A number of adult studies have shown that MTB DNA can be detected in sputum by Ultra (usually trace category positive) in subclinical (incipient) TB and also years after completion of “effective” antituberculosis therapy for prior pulmonary tuberculosis (17). To our knowledge there are no data on duration of Ultra positivity in children, but we do not feel this to be a plausible explanation in our study, as the median age of the children was 2.8 years (IQR, 1.3 to 5.3) and none of the children who were Ultra trace positive only had had prior PTB diagnosis or treatment. The possibility of false-positive results would also be more plausible for a child who was asymptomatic or from a low-prevalence area. In this study setting, all children had sufficient signs and symptoms predominantly for pulmonary TB to warrant initiation of anti-TB therapy by the study doctor. Moreover, the WHO Technical Expert Group recommended that among persons with HIV, children, and extrapulmonary specimens, “trace calls” should be considered to be true-positive results for use in clinical decisions and patient follow-up (17).

It is, however, curious that Ultra could be sensitive enough to pick up culture-negative TB but give negative results for a total of 13 samples that were positive by culture (LJ and/or MGIT) (Fig. 2). This finding could relate to sample processing. Samples were centrifuged, and then the cell pellet was resuspended in 2.5 ml PBS; unless the sample was well vortexed, creating a homogenous suspension, the bacilli could clump, and a greater number could be aliquoted into one test sample (the smear, Xpert, LJ culture, MGIT culture, or storage sample). Additionally, storing of an aliquot for a prolonged period (up to 2.5 years for early samples) could have negatively impacted the yield of the sample. However, on analysis of cases diagnosed by culture, 4/9 (44.4%) in 2016, 8/13 (61.5%) in 2017, and 1/9 (11.1%) in 2018 were discordant, suggesting no clear trend of discordance related to sample storage duration, although discordance was lower in more recent samples. Gastric lavage samples may perform less well than sputum on Ultra, as suggested by a recent study (18); the majority of samples in our study were gastric lavage fluid, and among the 13 Ultra-negative but culture-positive samples, 12 were gastric lavage fluid. Studies have documented inconsistent yields with different sample types collected for childhood TB diagnosis (19, 20), and this needs further investigation. However, the degree of discordance between DNA tests and culture highlights the fact that there remains a role for respiratory specimen cultures in children with suspected TB.

In light of the lack of a gold standard against which to evaluate the performance of Ultra, we used two reference standards, each susceptible to different forms of bias. Firstly, against the culture-based reference standard, Ultra has a higher sensitivity than Xpert MTB/RIF (58% versus 45%; P = 0.309 [nonsignificant]), and the specificity of Ultra was 94%, potentially relating to misclassification bias. A similar-sized study on stored samples from children in Cape Town yielded comparable sensitivity results against a culture-based reference (sensitivities of 74% for Ultra and 64% for Xpert, and a specificity of 97.2% for Ultra), adding weight to the validity of our findings, though they had fewer cases that were positive only by Ultra (n = 8).

The second reference standard used, the composite microbiological reference standard, risks incorporation bias by including the index test (Ultra) in the reference standard. Against a composite microbiological reference standard, the sensitivity of Ultra was superior to that of Xpert (72% versus 32%; P < 0.001) and LJ culture (72% versus 43%; P = 0.003) and nonsignificantly better than that of MGIT (72% versus 64%; P = 0.389).

The lack of clarity about the status of the trace-positive Ultra samples needs further investigation; we plan to do this by using data from all 1,204 children in the trial, including baseline clinical, radiological, and microbiologic data, when these are available for analysis at the end of the trial.

Ultra’s diagnostic sensitivity and ability to detect rifampin resistance, its turnaround time of 84 min, and the widespread availability of the Xpert platform in the public sector in low- and middle-income countries provide support for Ultra potentially being a best initial test for childhood PTB in such settings. Many countries are now transitioning from Xpert to Ultra, and the Xpert platform had been rolled out in 130/145 countries eligible for concessional pricing by 2016. WHO and the Foundation for Innovative New Diagnostics (FIND) have analyzed existing evidence and believe that for children and people living with HIV, the benefits of Ultra’s increased sensitivity outweigh the potential harm and cost of decreased specificity. It is interesting that our study showed that performing Ultra followed by liquid culture by MGIT identified 95% (49/50) of pediatric TB cases, which further indicates the value of liquid over solid culture in childhood TB diagnosis.

Strengths of this study include the large sample size, making it one of the larger studies of Ultra in childhood PTB to date, although the findings cannot necessarily be generalized outside the population in which it was studied, (i.e., those with minimal TB disease). Ultra may perform differently in different populations. All diagnostic testing for this trial was conducted in a College of American Pathologists (CAP) ISO 15189-accredited BSL-3 mycobacteriology laboratory, so the possibility of sample contamination or processing errors is potentially reduced. Limitations include the lack of baseline clinical data and outcome data for those who tested positive only with Ultra, as these data have yet to be finalized with completion of the SHINE trial. Including Ultra in the composite microbiological reference standard risks inclusion bias; however, we feel that this is justifiable, as Ultra is a highly specific test in vitro (98.7%) and is currently being used as a WHO-endorsed standard diagnostic test in this population. In Ugandan children with a high baseline disease prevalence, symptoms consistent with PTB, and anti-TB treatment given, a positive DNA-based test is less likely to represent a false positive than in other populations. Ideally, in our opinion, samples positive only by Ultra could be further tested for presence of mRNA or 16S rRNA to further investigate the presence of replicating or live bacteria. Furthermore, prolonged storage of specimen in a freezer may have affected the performance of Ultra, and this requires further investigation.

In conclusion, this study found Ultra to be more sensitive than Xpert for the detection of M. tuberculosis in Ugandan children, which represents an important advance for a condition which has posed a diagnostic challenge for decades. Ultra is likely to be the best initial test for suspected childhood PTB, but there is a still a role for culture in testing those for whom one or more sputum samples are Ultra negative. Culture-based rifampin susceptibility also remains important in low-bacillary-load samples (trace category positive) for which Ultra is unable to determine the status of rifampin drug susceptibility and reports “indeterminate rifampin resistance.” Further prospective diagnostic studies in which Ultra is performed in real time and in settings with different TB burdens would provide further information about its diagnostic performance.