Background

Tuberculous meningitis (TBM) is difficult to diagnose promptly. The utility of the Xpert MTB/RIF test for the diagnosis of TBM remains unclear, and the effect of host- and sample-related factors on test performance is unknown. This study sought to evaluate the sensitivity and specificity of Xpert MTB/RIF for the diagnosis of TBM.

Methods and Findings

235 South-African patients with a meningeal-like illness were categorised as having definite (culture or Amplicor PCR positive), probable (anti-TBM treatment initiated but microbiological confirmation lacking), or non-TBM. Xpert MTB/RIF accuracy was evaluated using 1 ml of uncentrifuged and, when available, 3 ml of centrifuged cerebrospinal fluid (CSF). To evaluate the incremental value of MTB/RIF over a clinically based diagnosis, test accuracy was compared to a clinical score (CS) derived using basic clinical and laboratory information.

Of 204 evaluable patients (of whom 87% were HIV-infected), 59 had definite TBM, 64 probable TBM, and 81 non-TBM. Overall sensitivity and specificity (95% CI) were 62% (48%–75%) and 95% (87%–99%), respectively. The sensitivity of Xpert MTB/RIF was significantly better than that of smear microscopy (62% versus 12%; p = 0.001) and significantly better than that of the CS (62% versus 30%; p = 0.001; C statistic 85% [79%–92%]). Xpert MTB/RIF sensitivity was higher when centrifuged versus uncentrifuged samples were used (82% [62%–94%] versus 47% [31%–61%]; p = 0.004). The combination of CS and Xpert MTB/RIF (Xpert MTB/RIF performed if CS<8) performed as well as Xpert MTB/RIF alone but with a ~10% reduction in test usage. This overall pattern of results remained unchanged when the definite and probable TBM groups were combined. Xpert MTB/RIF was not useful in identifying TBM among HIV-uninfected individuals, although the sample was small. There was no evidence of PCR inhibition, and the limit of detection was ~80 colony forming units per millilitre. Study limitations included a predominantly HIV-infected cohort and the limited number of culture-positive CSF samples.

Conclusions

Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals from a tuberculosis-endemic setting, particularly when a centrifuged CSF pellet is used. Further studies are required to confirm these findings in different settings.

Discussion

Although the utility profile and accuracy of Xpert MTB/RIF has been well characterised in sputum samples, there are hardly any data to guide its utility and implementation for TBM. This is critical as the rollout of Xpert MTB/RIF means that quantitative PCR is now available in many high burden settings, and data are urgently required to guide appropriate and relevant usage of this technology in biological fluids other than sputum. That Xpert MTB/RIF performs poorly in fluids from some compartments, e.g., the pleural space, highlights the need for such data [27]. The key findings of this study were as follows: (1) Xpert MTB/RIF is likely a good rule-in test for the diagnosis of TBM in HIV-infected patients; (2) centrifugation of the sample improved sensitivity in this context to almost 80%; (3) among HIV-infected patients, Xpert MTB/RIF performed significantly better than the widely available same-day alternative tests, i.e., smear microscopy, which suggests that prompt diagnosis of TBM is potentially achievable in the majority of patients in this setting; (4) the diagnostic value of Xpert MTB/RIF for HIV-infected patients is clinically meaningful given that it performed significantly better than hypothetical decision-making based on clinical characteristics and basic laboratory data (the CS); and (5) when combined with the CS, Xpert MTB/RIF test usage could be reduced by only a modest ~10% whilst retaining similar sensitivity and specificity compared to using Xpert MTB/RIF alone. This last finding informs clinical practice in resource-poor settings. Finally, we quantified the limit of detection of the assay, its relationship to bacterial load, and the impact of PCR inhibition. These data require reproduction in HIV-uninfected and non-TB-endemic populations.

There are limited data about Xpert MTB/RIF performance in TBM [28]. Published data include only small numbers of microbiologically proven TBM cases (range of 0 to 23) [29]–[32], often in a case-control design with a non-uniform reference standard, and often CSF-associated data were published as part of a laboratory-based evaluation of extrapulmonary TB samples, usually including samples from countries with low TB prevalence. Furthermore, there are no studies from high burden settings, and technical performance evaluations, including bacterial load studies, threshold level of detection, and impact of PCR inhibition, have hitherto not been undertaken.

Xpert MTB/RIF sensitivity was as high as 80% when a centrifuged CSF sample from an HIV-infected patient was used. This suggests that Xpert MTB/RIF, at least in an HIV-endemic environment, represents a possible new standard of care for the diagnosis of TBM. Sensitivity was considerably better than in previous studies using commercially available or non-standardised PCR tools [9],[32]–[34]. The ostensibly better performance is likely related to a combination of centrifugation (and hence concentration of bacilli) and technical aspects, including a more efficient standardised extraction protocol, fractionation of mycobacteria by a pre-sonication step, and a nested PCR protocol, thus maximising amplification. However, possibly higher bacterial loads in HIV-infected patients may have also played a role. Our findings have practical relevance because they imply that at least 3 ml of CSF should be set aside and centrifuged, and re-suspended in phosphate-buffered saline, before being run on the Xpert MTB/RIF. This high-sensitivity and potentially rapid diagnosis in most cases is likely to benefit HIV-infected patients suspected of having M.tb., as diagnostic and treatment delay is associated with higher mortality [35]–[37]. Impact-related studies are now required to verify this hypothesis. It is noteworthy that a second sample improved sensitivity minimally. These data suggest that, at least in an HIV-endemic setting, using a second cartridge is unlikely to give further benefit. However, larger studies are required to confirm this possibility.

Similar to the findings when using sputum, the level of detection of Xpert MTB/RIF was between 80 and 100 colony forming units per millilitre. This explains the sub-optimal sensitivity of Xpert MTB/RIF compared to culture, where the detection threshold is as low as 1–10 organisms per millilitre [38]. We did not find a correlation between TTP and Xpert MTB/RIF C_T values, as has been shown in sputum [39]. In contrast to previous PCR-based studies [40],[41], we found that CSF had a minimal inhibitory effect on the PCR reaction when compared to sputum. This may be due to the wash step incorporated into the assay that removes extracellular debris. We did not find a difference in TTP between the Xpert MTB/RIF–positive samples from centrifuged versus uncentrifuged CSF. This may be due to a type two statistical error, as the sample numbers were small.

There were three patients who were culture negative but Xpert MTB/RIF positive, i.e., Xpert MTB/RIF positive in the non-TB group. Our previous work has shown that such cases (Xpert MTB/RIF positive but culture negative) are likely to be true TB positives, and this is corroborated by high specificity obtained in large sputum-based studies where a significant minority of the patients had had previous TB [11]. If these culture-negative Xpert MTB/RIF–positive individuals are hypothetically designated definite-TB cases, then the overall case detection rate improves by a further ~10%.

The proper and meaningful value of a test lies in its ability to influence patient management through its incremental value over pre-test probability, or to have an impact on decision-making based on logical clinical judgement (based upon clinical features and basic laboratory parameters). We therefore derived a CS, hitherto unavailable for HIV-endemic settings, to evaluate Xpert MTB/RIF utility in clinical practice. Xpert MTB/RIF had significantly better performance outcomes than the clinical prediction rule (using a rule-in cut point, so appropriate comparisons could be made). Furthermore, hypothetically combining the CS with Xpert MTB/RIF resulted only in a modest ~10% reduction in test usage, but still maintained high sensitivity and specificity. These data suggest that clinical algorithms or scoring systems to limit test usage are unlikely to be significantly useful in resource-poor settings.

There are several limitations of our study. We could not determine the impact of Xpert MTB/RIF (time and proportion of patients initiated on treatment) compared to a smear microscopy/empiric treatment-based strategy given our study design and the fact that management decisions were not based on Xpert MTB/RIF results. However, this was because Xpert MTB/RIF had not yet been endorsed by the World Health Organization when the study commenced, had not been validated for use in CSF, and had been used as a research tool only (thus, for ethical reasons, study samples were evaluated only several weeks later). Although the confidence intervals of some of our estimates are wide (because of limited sample numbers), this is to our knowledge the largest diagnostic study undertaken in TBM (based on the number of microbiologically proven TBM cases; n = 59). This reflects the challenge and difficulty in performing such studies in resource-poor settings. It is possible that the Xpert MTB/RIF performs much better in HIV-infected individuals because of a possibly higher bacterial load, and thus our findings need to be confirmed in other settings. Given the small number of HIV-uninfected patients, we were unable to meaningfully compare this sub-group. The CS was developed to assess only incremental value above basic clinical and CSF parameters. The CS and the combination of CS plus Xpert MTB/RIF need prospective and independent validation. The non-significant difference in sensitivity between the paired centrifuged and non-centrifuged samples may reflect a type two statistical error, as the number of culture-positive paired samples was limited. Lastly, there were nine patients who could not be categorised within our defined groups and were excluded from the analysis.

In conclusion, Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals in a TB-endemic setting, particularly when a centrifuged CSF pellet is used. A second Xpert MTB/RIF test had minimal incremental benefit. Smear microscopy and the CS, when combined with Xpert MTB/RIF, only modestly minimised test usage in a resource-poor setting. Further studies are now required in non-HIV-endemic settings, and using validated scoring systems, to evaluate the impact of Xpert MTB/RIF on diagnostic accuracy, and morbidity and mortality in patients with TBM.

Source:PLOS