The performance of the new WHO-endorsed, Xpert MTB/RIF assay has recently been reported for some types of extra-pulmonary TB such as TB lymphadenitis , pleural TB , and TB meningitis . However, there are no comprehensive data about TBP to guide clinical practice. Here we report on the first large comprehensive study of Xpert MTB/RIF for the diagnosis of pericardial TB [5, 10]. It is also the first study to compare Xpert MTB/RIF to several alternative diagnostic assays, including ADA and IFN-γ, and to evaluate test performance outcomes in a TB and HIV-endemic setting.
The key findings of our study are that: (1) uIFNγ offers superior accuracy for the diagnosis of microbiologically confirmed TBP compared to the new Xpert MTB/RIF test and the established ADA assay; (2) PF Xpert MTB/RIF could bacteriologically confirm a TB diagnosis (and allow for drug susceptibility testing) in two thirds of patients with suspected TBP; (3) PF uIFNγ offered better rule-in diagnostic utility compared to ADA in current clinical use, while both tests could rapidly rule-out TBP; (4) PF Xpert MTB/RIF, when combined with either ADA or uIFNγ, offers >97% sensitivity and specificity for TBP diagnosis; and (5) concentration of PF samples prior to Xpert MTB/RIF testing increased the number of ‘indeterminate’ tests without significantly improving diagnostic yield.
Xpert MTB/RIF testing is undergoing phased implementation in a number of high burden settings for routine diagnosis of pulmonary TB [26, 27]. There is limited information on the diagnostic utility of the test in extrapulmonary cases of TB, and, in particular, Xpert MTB/RIF performance has only been evaluated in a very small number of PF samples . Our study is the largest systematic evaluation to date, and the first to examine Xpert MTB/RIF level of detection in PF and explore the effects of concentrating larger volumes of PF on Xpert MTB/RIF performance. Importantly, Xpert MTB/RIF testing could microbiologically confirm TB and allow drug susceptibility testing in almost two thirds of culture-positive cases, which is higher than in other body cavity fluids, including pleural, non-sputum biological fluids such as urine, and similar to performance in induced sputum specimens [13, 28, 29]. Preliminary level of detection experiments suggest that the Xpert MTB/RIF assay could reliably detect PF samples spiked with ≥75 cfus/ml of H37Rv, which is lower than the 131 cfu/ml limit of detection found in spiked sputum samples . Further studies with more replicates are required to confirm this finding. However, the diagnostic yield from PF was not improved by centrifugation of larger volumes and concentration only increased the number of ‘indeterminate’ test results, although this was not the result of an increase in PCR inhibition. The increased error rate may have resulted from reaction failure secondary to large amounts of pelleted blood and other inflammatory proteins found in pericardial exudates. Methods to further digest these proteins or the addition of a PCR-friendly blood lysis buffer may help to decrease error rates [31, 32]. Interestingly, unlike in sputum and pleural samples, no correlation was found between Xpert MTB/RIF-generated CT-values and liquid culture time-to-positivity using PF . However, the sensitivity of Xpert MTB/RIF was found to be significantly higher in HIV-positive versus negative patients, and this was due to the higher bacillary loads, as measure by liquid culture time-to-positivity (TTP), found in the PF of HIV-positive versus -negative TBP. This sensitivity difference may impact on the utility of Xpert MTB/RIF in low HIV prevalence settings.
Proof-of-principle studies in TB pericarditis have demonstrated the potential utility of using uIFNγ PF levels for diagnosis of TB pericarditis [6, 14, 34]. Although it can be easily measured, it is not routinely performed due to its high cost and the kits only being available in a 96-well format, which would lead to a considerable wastage of unused wells [5, 35]. However, the recent availability of a low-cost assay (Intergam, Antrum Biotech, Cape Town, South Africa) which is tested in this study may allow for more widespread use of uIFNγ for the diagnosis of TBP in clinical practice. In this study, using ROC-curve analysis, we demonstrate an optimal cut-point of 44 pg/ml, and show that with this cut-point of uIFNγ we could detect almost all definite-TB cases (missing only three cases) and incorrectly classified only one non-TB case.
Are the findings of this study generalisable to other settings, and does either Xpert MTB/RIF or uIFNγ testing potentially offer utility beyond existing same-day diagnostic tools, such as smear microscopy, PF ADA measurements and/or basic clinical information? In this study we compare the utility of Xpert MTB/RIF, uIFNγ or ADA, alone or in combination across different TB prevalence rates, focusing on the diagnostic priorities of rapid rule-in and rule-out, as well as bacteriologically confirmed diagnosis. In a high prevalence setting (TB prevalence >30%), Xpert MTB/RIF and uIFNγ outperforms ADA and basic clinical predictors for rapid rule-in (highest LR + and PPV). However, both ADA and uIFNγ offer equivalent rapid rule-out utility, outperforming Xpert MTB/RIF and clinical predictors. Combining Xpert MTB/RIF testing followed by ADA or uIFNγ in Xpert-negative PF maximised both sensitivity and specificity to >97% for TBP diagnosis. This may offer the best diagnostic approach in high burden settings, especially where drug-susceptibility testing is desirable, but the cost of a two test algorithm will remain a key consideration in resource-poor conditions where TB is endemic. Xpert MTB/RIF currently costs approximately US$20/test, while ADA measurement is less than US$0.1/test. Intergam kits are not currently commercially available so the cost is unknown but likely to be only slightly more than smear microscopy. Prospective studies of the cost-effectiveness of diagnostic options are needed before it can be considered for clinical practice.
Our study had a number of important limitations. This study did not optimise PF sample volumes or processing beyond comparing two volumes and a simple centrifugation step thought applicable to resource-limited settings. The use of different volumes or alternative processing methods may have improved Xpert MTB/RIF sensitivity and/or decreased the high indeterminate rate found. A low number of replicates were performed in limit of detection experiments and these findings should be confirmed in further studies. The study was conducted in a high TB and HIV burden setting, which may limit the generalisability of the findings. Performance may differ in a low TB burden setting and where HIV co-infection rates and, hence, bacterial load, are lower, such as Europe and the US. However, the use of diagnostic accuracy measures that are less affected by prevalence, such as LRs, and generating estimates across varying TB prevalence rates helps to highlight potential performance differences between low and high burden settings and, hence, improve generalisability. Whilst this is the largest study that has comprehensively evaluated several diagnostic strategies and tools in the same prospective cohort, the sample size was limited in the non-TB group. The small number of non-TB patients reflects the high burden of infectious and HIV-related disease in the South African environment . Although the use of a combined reference standard may introduce a minor degree of selection bias, this consideration is outweighed by the avoidance of misclassification bias when using a culture only reference (data provided in the online supplementary materials).