Can improved diagnostics reduce mortality from Tuberculous

Tuberculous meningitis (TBM) is the second most common Background: cause of meningitis in sub-Saharan Africa and is notoriously difficult to diagnose. We describe the impact of improved TBM diagnostics over 6.5 years at two Ugandan referral hospitals. Cohort one received cerebrospinal fluid (CSF) smear microscopy Methods: only (2010-2013). Cohort two received smear microscopy and Xpert MTB/Rif (Xpert) on 1ml unprocessed CSF at physician discretion (2011-2013). Cohort three received smear microscopy, routine liquid-media culture and Xpert on large volume centrifuged CSF (2013-2017) for all meningitis suspects with a negative CSF cryptococcal antigen. We compared rates of microbiologically confirmed TBM and hospital outcomes over time. 1672 HIV-infected adults presenting with suspected meningitis Results: underwent lumbar puncture, of which 33% (558/1672) had negative CSF cryptococcal antigen and 12% (195/1672) were treated for TB meningitis. Over the study period, microbiological confirmation of TBM increased from 3% to 41% (P<0.01) and there was a decline in in-hospital mortality from 57% to 41% (P=0.27) amongst those with a known outcome. Adjusting for definite TBM diagnosis and antiretroviral therapy use, and using imputed data, assuming 50% of those with an unknown outcome died, the odds of dying were nearly twice as high in cohort one (adjusted odds ratio 1.7, 95% CI 0.7 to 4.4) compared to cohort three.  Sensitivity of Xpert was 63% (38/60) and culture was 65% (39/60) against a composite reference standard. 1,2 3 4 5 6 7 8 2,8 2 6 1,9 2,10

was 65% (39/60) against a composite reference standard. As TBM diagnostics have improved, microbiologically-confirmed Conclusions: TBM diagnoses have increased and in-hospital mortality has declined. Yet, mortality due to TB meningitis remains unacceptably high and further measures are needed to improve outcomes from TBM in Uganda.

Introduction
Tuberculous meningitis (TBM) is the second most common cause of adult meningitis in sub-Saharan Africa 1,2 , accounting for one to five percent of the 10.4 million tuberculosis (TB) cases reported worldwide in 2016 3 . Despite treatment, TBM outcomes are poor with 19-28% mortality in HIV-uninfected persons and 40-67% mortality in HIV-infected patients in addition to long-term disability is frequent among survivors 4-6 .
Insidious symptom onset in persons with TBM leads to delay in seeking care and increasing disease severity at presentation correlates with higher mortality 7 . Further, the paucibacillary nature of TBM increases the difficulty in confirming diagnosis once care is sought, also contributing to high mortality 8 . Cerebrospinal fluid (CSF) smear microscopy for acid-fast bacilli (AFB smear) has poor sensitivity (~10-20%) in routine practice 7 . Culture has improved sensitivity (~50-60%) but is not widely available in many resource constrained settings and commonly takes at least 2-3 weeks for liquid culture growth, which is too slow to guide decision-making at the time of presentation 8 .
In 2013, the World Health Organization endorsed the Xpert MTB/RIF (Xpert) assay (Cepheid, Sunnyvale, California, USA), a cartridge-based, polymerase chain reaction assay with a run time of 113 minutes, as the preferred initial test to investigate TB meningitis on the basis of a meta-analysis of 13 studies 9 .
Of the two major studies included in the meta-analysis, Patel and colleagues reported 67% sensitivity against microbiologically proven TBM and 36% against consensus clinical case definitions, while Nhu and colleagues showed 59% sensitivity against the same case definitions 10-12 . Additionally, use of a larger volume of centrifuged CSF improves sensitivity of Xpert 10,13 . Yet, inadequate negative predictive value means that Xpert cannot substitute for clinical judgement 14 .
There is evidence that use of Xpert for diagnosis of pulmonary TB reduces diagnostic delay, increases the rate of same day treatment, and decreases usage of empiric treatment 15,16 . However for pulmonary TB, Xpert has not been shown to decrease mortality [16][17][18] . Yet, lessening diagnostic delay in persons with TBM may be more likely to lead to improved outcomes as compared to pulmonary TB given the high early mortality of TBM 19 . Whether routine use of Xpert for investigation of suspected TBM has made an impact on mortality has not yet been investigated.
Herein we describe TBM diagnosis and outcomes over a 6.5-year period in prospective cohorts at two Ugandan referral hospitals.

Assessment of outcome
In-hospital outcome was determined from case report forms, hospital medical records or follow-up telephone calls with the patient or their surrogate where hospital outcome was unknown. The outcome was categorised as discharged alive, deceased prior to hospital discharge or unknown (i.e. self-discharged against medical advice in an imminently terminal patient, hospital outcome undetermined, transferred to another facility).

Statistical methods
Comparisons of categorical and continuous demographic and clinical characteristics by cohort were performed using Fisher's  Mortality was first compared by cohort for participants with a known outcome using Fisher's exact test. Data for patients with unknown outcome was imputed to assume first that 50% within each cohort died, or that 75% died (both within the expected mortality range for this population). Odds ratios and 95% confidence intervals were computed from multivariable logistic regression models with these imputed data, adjusted for 1) ART status, and 2) ART status and definite TBM diagnosis. Imputations were repeated with new random assignments to confirm results. Analyses were conducted using SAS version 9.4 (The SAS Institute, Cary, NC) and p-values <0.05 were considered statistically significant.  Figure 2). Neither method diagnosed significantly more cases than the other (p=0.42).

Outcomes
Hospital outcome was known for 142 participants, 53 had unknown outcomes or self-discharged against medical advice.
Median time to death was 3 days (IQR 1-9 days) among those known to have died, and median length of hospitalization was 7 days (IQR 4-10 days) for participants known to have survived to hospital discharge. Among those with known outcomes, there was a non-significant decline in mortality from 57% in cohort one to 41% in cohort three (p=0.27) (  Figure 3).

Discussion
Rapid molecular diagnostics have been predicted to reduce TB-related mortality 21 but no prior studies have looked at the impact of Xpert on TBM-related mortality. Here we report clinical outcomes among hospitalized Ugandans treated for TB meningitis over a 6.5-year period. In-hospital mortality   2.0 (0.6,6.7) 1 Overall median (IQR) time in hospital was 7 (4, 10) days among those who were known to be discharged alive, and 3 (1, 9) days among those who were known to have died in hospital *P-value from Fisher's exact test comparing KNOWN discharged alive vs KNOWN died; Odds ratios are the odds of being discharged alive, assuming 50% and 75% of those with unknown outcome died was high in the cohort overall (44%), similar to other research settings with high HIV prevalence 7, 19,22,23 . The adjusted model on imputed data found odds of in-hospital mortality were almost two fold higher in the earliest cohort, tested by CSF smear microscopy only, than that of that of the most recent cohort in whom Xpert (and culture) are routinely performed. Severity of TBM at presentation was similar over the study period and TBM treatment recommendations have not changed for Uganda, though other confounding factors may exist.
Although we lack data regarding time to treatment initiation, in this research setting Xpert results were obtained within 24 hours, leading to prompt treatment in the 51 subjects who were positive by Xpert MTB/Rif in real-time and presumably contributing to reduced mortality. The proportion of ART experienced subjects increased significantly over time with the roll out of ART treatment in Uganda and because the parent trial in cohort one enrolled only ART naïve subjects 24 . Although ART status was not associated with mortality, we did adjust for ART in multivariable models due to the large discrepancy in ART status between cohorts.
Despite a non-significant decline in mortality, a current casefatality rate of 41% remains unacceptably high and highlights the remaining work required to achieve the WHO goal of reducing TB-related deaths by 90% by 2030 25 . Initiating treatment in the early stage of disease is the single most important factor in improving outcomes 7 . Earlier presentation to the hospital is essential for prompt diagnosis and treatment initiation, yet, 83% of our cohort presented with MRC grade II or III disease.
Once the patient presents to care, an affordable, rapid, and reliable test that can effectively confirm or rule out TBM is crucial for prompt diagnosis. In this predominantly HIV-positive TBM cohort, sensitivity of Xpert was 63% against the composite reference standard. Thus, even though results were available rapidly, Xpert missed over one in three cases. The next generation assay Xpert MTB/Rif Ultra has an analytic limit of detection of 15 colony forming units (CFU)/ml, compared to 113 CFU/ml for Xpert 26 . Ultra appears to be significantly more sensitive than Xpert or culture for the diagnosis of TBM (95% versus 45% and 45% respectively, P<0.001) 27 . Whether Ultra can reduce diagnostic delay and improve outcome from TBM requires further prospective evaluation.
Where both Xpert and MGIT had been done, less than a third (23%, 17/74) of confirmed cases were positive by both modalities. This is consistent with prior findings and is likely due to the relatively higher sensitivity of culture versus Xpert, and the ability of Xpert to detect dead TB bacilli 13,27 . Neither test performed better than the other (P=0.42). Figure 3. Illustration of odds of dying in cohort one and two compared to cohort three in a multivariate model. Odds ratios (and 95% confidence intervals) for death by the end of hospitalization comparing cohorts 1 and 2 to cohort 3, computed from multivariable logistic regression models with imputed data, adjusted for (1) ART status, and (2) ART status and definite TBM diagnosis. Data for patients with unknown outcome was imputed to assume that 50% within each cohort died, or that 75% died.
Until a highly sensitive assay is widely available, there is likely to be on-going heterogeneity in clinical practice regarding initiation of empiric therapy for TBM. In our study, Mulago Hospital participants were treated for TBM on an empiric basis in under one quarter of cases as opposed to over three quarters of cases at Mbarara Hospital. Though empiric TBM therapy is potentially life-saving, significant risks such as side effects, drug-interactions and adjunctive steroids in an already immunosuppressed population need to be considered. Ideally, a rapid, accurate test allows therapy for TB meningitis to be started promptly only in those who actually have TBM. Overall, the proportion with microbiologically confirmed TBM increased significantly from 3% in cohort one to 41% in cohort three (P<0.01). In cohort two, Xpert was only performed in cases where there was extremely high index of suspicion and empiric treatment was given only twice in those with a negative Xpert (4%, 2/56). The low number of empiric diagnoses during this period were likely due to over-confidence in Xpert's ability to rule-out TBM. As understanding regarding the limitations of Xpert for the diagnosis of TBM became known, empiric TBM treatment rose 14 .
Limitations of this study include missing data on hospital outcomes and time to starting TB treatment, unbalanced numbers in each cohort including smaller numbers in earlier cohorts and lack of long-term outcome data. When imputing data in the model we assumed that either 50% or 75% of patients with unknown outcome actually died, which is a clinically reasonable judgment for this population 4 .
Here we present important data on rates of diagnostic confirmation and TBM mortality during a period of TB diagnostic evolution. There has been a significant increase in microbiological confirmation and a modest, albeit non-significant, decline in mortality since introduction of Xpert and culture in our study setting. An on-going multifaceted approach is needed to further reduce death and disability from TBM.

Consent
Written informed consent for publication of the anonymised data was obtained from the participants or their surrogates.

Data availability
The database contains individual level data and as such is not available through an open-access data repository. The database is stored on a secure server at University of Minnesota. Researchers interested in accessing the data can contact the corresponding author (FVC), the last author (DRB) or the Division of Biostatistics at the University of Minnesota. Data access will be granted to active researchers in the field with the agreement of the authors.

Competing interests
No competing interests were disclosed. Thank you for the opportunity to review this article which describes the investigation, treatment and outcomes of a cohort of mostly HIV infected adults in Uganda who were suspected of having TB meningitis. It is well written and easily reproducible by others.

Grant information
The title asks the question of whether improved diagnostics can reduce mortality in TBM. It might be better to say 'Have improved diagnostics improved outcomes?' My view of the data is that it provides very limited evidence that improved diagnostics have improved outcomes of TBM in this cohort. The small improvements in outcome over time are statistically non-significant and due to the observational design are likely to be influenced by numerous unmeasured confounding factors. For example, the experience of clinician may well have changed over time, we know that empiric treatment was used differently in the 2 centres but it might also have changed over time. Part of the work-up of a patient with suspected TBM is a search for extra-neural TB and we do not know how this changed over time although it is likely that Xpert MTB/RIF was used for non-CSF samples in later cohorts compared to earlier, the same might be said for urine LAM.
My view as a reader is therefore that this data does not provide convincing evidence that the change in diagnostics was the driver of the small changes in mortality. It must be remembered that new diagnostics also have the potential to worsen outcomes, particularly if clinicians miss-interpret negative tests as ruling out the condition as might well occur with Xpert MTB/RIF and TBM.
I think that this well written work definitely deserves to be published but feel that the conclusions should reflect a greater level of uncertainty.

Minor points:
The abstract says that all 1672 patients were HIV infected but in the results section is says 96% were HIV infected so there is a discrepancy.
Last line, para 1 of introduction, probably remove word 'to'.
Last line. Para 3 of introduction-It is not that Xpert cannot substitute for clinical judgement-Xpert is used to enhance clinical judgement, it's just that the poor sensitivity means that when negative it has limited influence on decision making Statistical methods-the uniform case definition is probable or definite TBM-do the authors think this is a Statistical methods-the uniform case definition is probable or definite TBM-do the authors think this is a reasonable reference standard for other tests? My view is that the threshold for treating TBM is very low and that patients with possible/probably or definite TBM should receive treatment and therefore this would be a more appropriate reference standard-what are your views on that?
Methods-Not clear if there were 76 or 74 microbiologically proven cases Discussion-Para 1, repeat of 'that of' Discussion-Para 4, First sentence implies that tests are the only answer to the diagnosis of TBM, what about clinical prediction rules relying on clinical data-although so far they have not been very successful, neither have tests so a robust CPR may negate the need for tests.
Discussion-Para5, Do the authors really think there are dead bacilli in CSF? Some of the authors have argued the opposite in their recent paper on Xpert Ultra so you can't have it both ways.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
Referee Expertise: Clinical management of patients with HIV and meningitis. The interpretation of tests and thresholds for treatment.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.