Tuberculous meningitis

Diagnostic Accuracy of Quantitative PCR (Xpert MTB/RIF) for Tuberculous Meningitis in a High Burden Setting: A Prospective Study.

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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 CT 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

Utility of the Xpert MTB/RIF Assay for Diagnosis of Tuberculous Meningitis.

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Tuberculous meningitis (TBM) is characterized by copious cerebrospinal fluid (CSF) inflammation and yet few Mycobacterium tuberculosis. This combination creates a disease that is notoriously difficult to definitively diagnose. In this week’s issue of PLOS Medicine, Patel and colleagues report the diagnostic performance of the GeneXpert system’s Xpert MTB/RIF assay for the diagnosis of TBM in a cohort of 204 South African, predominantly HIV-infected, adults presenting with suspected meningitis of whom 59 had definitive TBM [1]. The Xpert MTB/RIF assay’s overall sensitivity was 62%, and specificity was 95%. The performance was better using larger volumes of centrifuged CSF among HIV-infected persons with sensitivity of approximately 80% and excellent specificity for microbiologically confirmed TBM. Xpert MTB/RIF performance was less impressive using uncentrifuged CSF with a sensitivity of ≤50%, and Xpert MTB/RIF performance was negligible in HIV-uninfected persons.

What Is GeneXpert?

The GeneXpert System (Cepheid) is a single use cartridge-based real-time PCR fully automated system that performs sample decontamination, sonication, automated nucleic acid amplification, and fluorescence-based quantitative PCR [2]–[4]. The Xpert MTB/RIF assay, developed by David Alland, detects M. tuberculosis DNA in approximately 2 hours with minimal hands-on time[3]. This new technology was endorsed by the World Health Organization in December 2010, and as of June 30, 2013, a total of 1,402 GeneXpert instruments and over 3 million Xpert MTB/RIF cartridges have been procured in the public sector in 88 countries [5]. The concessional pricing is US$9.98 per cartridge for 145 low- and middle-income countries [6]. The same GeneXpert platform also can be used for a variety of US Food and Drug Administration (FDA)-approved testing (e.g., influenza, Clostridium difficile, methicillin-resistant Staphylococcus aureus).

What Is the Performance of Xpert MTB/RIF?

There is a rapidly emerging literature regarding the performance of the Xpert MTB/RIF assay. Fundamentally, the sensitivity depends on the burden of organisms and thereby the target DNA present in the specimen. The published Xpert MTB/RIF detection threshold is approximately 100–130 colony forming units (cfu)/ml of sample [2],[3]. Patel and colleagues observed a similar threshold of 80–100 cfu/ml of CSF in this study [1]. In comparison, the detection threshold is <10 cfu/ml for mycobacterial liquid culture and is >5,000 cfu/ml for Ziehl-Neelsen staining for acid fast bacilli (AFB) via standard microscopy in sputum [7]–[9]. In real world clinical terms, this means 98%–99% detection by Xpert MTB/RIF of AFB smear-positive pulmonary TB, and approximately 75% detection of smear-negative, culture-positive pulmonary TB [3],[10],[11].

The threshold of detection is a key principle. The Xpert MTB/RIF test performs better when there is a larger burden of infectious organisms present in the specimen being tested. Yet, TB meningitis is a paucibacillary condition with few organisms. More organisms are likely present when the host is immunocompromised, or when a larger input volume is used for the test. Thus specimen centrifugation can compensate and should improve diagnostic yield, as demonstrated by a 35% improvement in sensitivity in this study [1].

The prior data on Xpert MTB/RIF testing of CSF are limited. In India, the Xpert MTB/RIF assay detected two of seven culture-positive specimens using an input volume of ~1 ml [12]. In an Italian study, 11 of 13 TBM patients were detected by Xpert MTB/RIF using an input volume of 2 ml into the cartridge without the standard N-acetyl-L-cysteine-sodium hydroxide (1% NALC-NaOH) decontamination and mucolytic step (i.e., GeneXpert Sample Reagent) [13]. This sample reagent was designed for sputum samples. Numerous commercially available PCR assays exist for other pathogens (e.g., herpes simplex PCR) without such a decontamination step [13], and the necessity of using the sample reagent for non-bloody CSF is unclear.

Public Health Significance

Although two commercial TB PCR tests previously existed [14], the innovation is that the GeneXpert platform is fully automated and is being rolled out in low- and middle-income countries. Thus, GeneXpert is an actual technology that can be—and is being—widely used globally. However, immediate implementation of a US$10 Xpert MTB/RIF assay for all cases of meningitis is unwise and unsustainable. Further research is needed on how best to incorporate the Xpert MTB/RIF test into diagnostic testing for meningitis, to ensure that it is a cost-effective intervention that improves health and does not waste resources.

Patel and colleagues modeled a clinical score to predict who had such high pretest probability of TBM that Xpert MTB/RIF was unnecessary to perform [1]. Yet health systems also need the opposite, a clinical score or algorithm to identify who has such low pretest probability that they do not require testing. Several investigators have developed meningitis diagnostic algorithms, yet broader validation is needed [15]–[17]. Ordering comprehensive testing of all the available diagnostic tests for every patient with suspected meningitis, including Xpert MTB/RIF testing, is 3–4-fold more expensive, without any better diagnostic yield than a targeted stepwise approach[15].

Key Principles of TB Meningitis Diagnosis

Despite molecular diagnostics, there remain a number of key pieces of information that inform clinicians as to the likelihood of a TBM diagnosis, so as to target testing in a cost-effective manner. The first is history. TBM is a subacute illness. Symptoms <6 days are atypical for TBM, yet near universal for bacterial meningitis [16]. Second is the immunology of the patient. Immunosuppression due to HIV/AIDS or age (e.g., infants, elderly) are key drivers of TBM, and immunosuppression increases the bacillary burden of M. tuberculosis organisms, likely increasing the diagnostic yield of molecular testing. In the current study, the GeneXpert performed poorly among HIV-uninfected persons, and the performance in children is unknown. Third is the CSF profile. TBM is classically a lymphocytic meningitis (i.e., >30%–50% lymphocytes in >90% of persons [16],[18]); with a low CSF glucose of <60% of serum glucose or an absolute CSF glucose concentration <2·2 mmol/l (<40 mg/dl) in >92%–95% [18]–[20].

In HIV-infected adults, the clinical history and CSF profile overlap extensively with meningitis due to Cryptococcus neoformans, and cryptococcal meningitis is overall the most common meningitis etiology in adults in sub-Saharan Africa [15]. Thus before a US$10 Xpert MTB/RIF test is performed for a less common condition, a US$2 cryptococcal antigen lateral flow assay should likely be performed for a more frequent condition [15].

If there is insufficient CSF volume available for testing (i.e., ❤ ml), in a clinically stable patient treated presumptively for bacterial meningitis a repeated lumbar puncture in 48 hours is likely a better strategy than sub-optimal Xpert MTB/RIF testing using a limited volume. A repeat lumbar puncture can collect a sufficiently large volume as well as reassess CSF glucose. At 48 hours, the CSF glucose should have risen by >100% of the initial level in treated bacterial meningitis[16]. Persistently low CSF glucose levels at 48 hours coupled with excluding cryptococcal meningitis should prompt Xpert MTB/RIF testing and/or empiric anti-TB therapy [16].

Xpert MTB/RIF appears to be a highly useful test to “rule in” the diagnosis of TBM, yet the clinical acumen of physicians remains a necessity for the wise use of any new diagnostic test. Careful application of these new diagnostic tools should improve clinicians’ ability to deliver timely, cost-effective care to patients with suspected TBM throughout the world, an approach that future studies should systematically evaluate.

Source:PLOS

Rifampicin and moxifloxacin for tuberculous meningitis.

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Ravina Ruslami and colleagues presented a study assessing pharmacokinetics, safety, and survival benefit of different treatment regimens containing high-dose rifampicin and moxifloxacin in patients with tuberculous meningitis in a hospital setting.1

Their findings that a treatment regimen containing a higher dose of rifampicin and standard-dose or high-dose moxifloxacin during the first 2 weeks is safe in patients with tuberculous meningitis, and that high-dose intravenous rifampicin could be associated with a survival benefit in patients with severe disease are important to note.

We agree with the authors that, on the basis of the small number of patients per group, clinical results should be interpreted carefully. To compensate for small group sizes, one could consider a different strategy with drug exposure as a continuous variable. Additionally, isoniazid concentrations should also be measured since isoniazid contributes to rapid culture conversion and penetrates well in cerebrospinal fluid. Receiver operating characteristic analysis could show the extent to which cumulative drug exposures of rifampicin, moxifloxacin, and isoniazid relate to outcome. Resultant potentially crucial values for positive treatment outcome could be detected and related to the antagonistic effect on cell kill as observed after co-administration of rifampicin and moxifloxacin in in vitro and in vivo studies.2, 3

Another consideration is the potential benefit of a higher oral dosage to reach similar drug exposure as achieved with intravenous dosing. The proposed alternative strategy to analyse the data would also compensate for the difference in drug exposure due to intravenous administration compared with oral dosing, especially in the presence of predisposing factors for poor drug absorption like HIV co-infection.

We therefore would like to encourage the authors to do further analyses of their data to generate additional hypotheses.

References

1 Ruslami R, Ganiem AR, Dian S, et al. Intensified regimen containing rifampicin and moxifloxacin for tuberculous meningitis: an open-label, randomised controlled phase 2 trial. Lancet infect Dis 2013; 13: 27-35. Summary | Full Text | PDF(279KB) |CrossRef | PubMed

2 Drusano GL, Sgambati N, Eichas A, Brown DL, Kulawy R, Louie A. The combination of rifampin plus moxifloxacin is synergistic for suppression of resistance but antagonistic for cell kill of Mycobacterium tuberculosis as determined in a hollow-fiber infection model. mBio 2010; 1: e00139. e00110 PubMed

3 Balasubramanian V, Solapure S, Gaonkar S, et al. Effect of coadministration of moxifloxacin and rifampin onMycobacterium tuberculosis in a murine aerosol infection model. Antimicrob Agents Chemother 2012; 56: 3054-3057. PubMed

Source:Lancet