|Year : 2021 | Volume
| Issue : 4 | Page : 428-432
Tuberculostearic acid, a potential parameter for scoring system construction for tuberculous meningitis diagnosis
Tsz Hei Fong, Wangpan Shi, Siyi Li, Guanghui Liu, Chung Lam Ng, Haishan Jiang
Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, China
|Date of Submission||16-Aug-2021|
|Date of Decision||12-Sep-2021|
|Date of Acceptance||05-Oct-2021|
|Date of Web Publication||13-Dec-2021|
Department of Neurology, Nanfang Hospital, Southern Medical University, No. 1838 Guangzhou North Avenue, Guangzhou 510515
Source of Support: None, Conflict of Interest: None
Background: This study aimed to validate the value of tuberculostearic acid (TBSA) whether it could implicate the existence of Mycobacterium tuberculosis (Mtb) and assist for the clinical diagnosis of tuberculous meningitis (TBM). Methods: The patient's cerebrospinal fluid (CSF) specimen was collected through the lumbar puncture and detected for TBSA with gas chromatography/mass spectrometry. At the same time, gold standard tests, i.e., CSF direct culture, CSF smear microscopy, or nucleic acid amplification tests, for Mtb were routinely performed. Furthermore, we evaluated all patients by the Lancet consensus scoring system, which classifies suspected patients to “Definite (depend on gold standard results only),” “Probable (>10 pts without imaging or >12 pts with imaging information),” “Possible (6–9 pts without imaging or 6–11 pts with imaging),” and “Not (<6 pts or with alternative diagnoses)” TBM. Results: In total, 140 patients were admitted for our study included 27 confirmed TBM patients and 50 TBSA-positive patients. Sensitivity (0.7407, confidence interval [CI] 95%: 0.5372–0.8889) and specificity (0.7345, CI 95%: 0.6432–0.8132) were calculated. The Lancet consensus scoring system was also applied to evaluate the possibility of TBM in suspected patients, finding that TBSA-positive patients showed a similar grouping distribution as the definite TBM patients. Conclusions: Our study implicates that the prospective utilization of TBSA is worth combining into a scoring system for characterizing the features of Mtb, showing a great potential of TBM diagnosis by TBSA in future.
Keywords: Diagnosis, gas chromatography/mass spectrometry, tuberculostearic acid, tuberculous meningitis
|How to cite this article:|
Fong TH, Shi W, Li S, Liu G, Ng CL, Jiang H. Tuberculostearic acid, a potential parameter for scoring system construction for tuberculous meningitis diagnosis. Int J Mycobacteriol 2021;10:428-32
|How to cite this URL:|
Fong TH, Shi W, Li S, Liu G, Ng CL, Jiang H. Tuberculostearic acid, a potential parameter for scoring system construction for tuberculous meningitis diagnosis. Int J Mycobacteriol [serial online] 2021 [cited 2022 Jan 21];10:428-32. Available from: https://www.ijmyco.org/text.asp?2021/10/4/428/332346
| Introduction|| |
Tuberculous meningitis (TBM) is the most dreaded type of extrapulmonary tuberculosis that developed in the central nervous system (CNS), although the average incidence of TBM remains low in tuberculosis patients (5%~15%) as well as infectious meningitis (1%). However, the mortality of TBM is high and a study reported that the dosage of treatment, especially rifampicin, might not be related to the reduction of mortality in TBM patients. Hence, among the current options, early diagnosis ensuing timely treatment of TBM is the most significant strategy to reduce mortality and improve outcomes rather than simply optimizing the therapies.
However, one of the major issues concerning TBM is low sensitivity in cerebrospinal fluid (CSF) microscopy and molecular assays (i.e., Ziehl − Neelsen staining, nucleic acid amplification test (NAAT), and metagenomic next-generation sequencing) for early diagnosis, which complicates the clinical judgment and challenges the prevention of further neurologic damage. Direct CSF Mycobacterium tuberculosis (Mtb) culture, a more sensitive method, requires liquid and solid media culture for at least 10 days and up to 8 weeks, respectively, as well as, ideally, a laboratory with biosafety level three. Moreover, all these direct methods in identifying Mtb, adopted as gold standard, have limitations regarding the early diagnosis of TBM.
On another aspect, the Lancet consensus scoring system is a well-developed system for improving TBM diagnostic accuracy, incorporating various criteria generating from clinical features, CSF findings, as well as neurological imaging. Nevertheless, this system was not updated with some newly proposed diagnostic biomarkers for TBM, such as C-reaction protein, adenosine deaminase, procalcitonin, or etc., Furthermore, even though the parameters included in this scoring system were proved with a high correlation with TBM (e.g., cerebrovascular infarcts), limitations remain exist in treatment decisions.
Tuberculostearic acid (TBSA) is a specific cellular composition of fatty acid in mycobacteria and has been considered as a biomarker in Mtb detection from CSF specimen. Traunmüller et al. showed that TBSA in the plasma is valuable assisting diagnosis in active tuberculosis because it is reported that considerable amounts of TBSA are released into blood circulation in TB-infected patients. In terms of TBM, some studies showed that gas chromatography/mass spectrometry (GC/MS) technique is highly sensitive for detecting TBSA in CSF at the scale of picogram (10–12 g).,
In our study, we aim to verify whether TBSA in CSF is a sensitive marker for patients and a complementary diagnostic parameter for insensitive gold standard tests in TBM.
| Methods|| |
Patients and controls
In this study, we have admitted 140 patients (age >11 years) with the symptoms and signs of TBM including one or more of the following manifestations: headache, irritability, vomiting, fever, neck stiffness, convulsions, focal neurological deficits, altered consciousness, or lethargy. No patients were excluded and all of them were recruited and treated at Nanfang Hospital and Zhujiang Hospital in Guangzhou, China.
The patient's CSF specimen was collected through the lumbar puncture and tested for TBSA with GC/MS. CSF direct culture (LöwensteinJensen Slope Medium), CSF smear microscopy (Ziehl-Neelsen staining), or NAAT (real time-quantitative polymerase chain reaction using Mtb complex nucleic acid detection kit [Qiagen, Germany]) for Mtb were performed, which are known as the gold standard tests for TBM diagnosis, to distinguish the definite TBM from suspected patients. Cerebrospinal fluid of each patient would be examined by at least one of the three tests. One positive result in one of the three assays found in the suspected patients was determined as “positive” TBM. All the results of gold standard tests were provided by the Laboratory Medicine Center of Nanfang Hospital.
At the same time, we evaluated all patients by Lancet consensus scoring system that requires the presence of symptoms or signs indicative of TBM plus additional (1) clinical, (2) CSF, (3) cerebral imaging criteria, and (4) evidence of TB elsewhere. The four criteria contained a total of 20 parameters [Table 1] to score patients with different points of each parameter (maximum score: 20 pts). Suspected patients were classified by assigning scores into either “Definite (depend on gold standard results only),” “Probable (>10 pts without imaging or >12 pts with imaging information),” “Possible (6–9 pts without imaging or 6–11 pts with imaging),” or “Not (<6 pts or with alternative diagnoses)” TBM.
|Table 1: Parameters in the lancet consensus scoring system in Marais et al. (2010)|
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All patients were satisfied with the conditions of lumbar puncture and were informed consent for our research purpose and sample usage. The collected samples were kept in the CSF sample library of Department of Neurology in Nanfang Hospital. The study was approved by the Medical Ethics Committee of Nanfang Hospital (Number of approval letter: NFEC-2018-087).
Chemical pretreatment and chromatographic conditions
CSF specimens were collected by lumbar puncture under aseptic conditions and stored in sterile test tubes. All CSF specimens were centrifuged (Centrifuge: Sorvall ST 16R, Thermo Fisher Scientific, USA) at 1006.2 × g for 10 min at 4°C and 500 μl of the supernatant was dispensed into a sterile cryotube, rapidly condensed in liquid nitrogen, and stored at −80°C. 100 μl of CSF from collected specimens were used for TBSA detection and which was mixed with 300 μl of methanol. 50 μl of internal standard (0.3 g/mL Nonadecanoic-d 37 acid) was added. After 15 s of vortexing, the mixture was centrifugated for 15 min at 25,155 × g. 370 μl of the supernatant was transferred to a new EP tube and dried by blowing nitrogen at the room temperature. Then, 50 μl of 20 mg/mL methoxyamine pyridine solution and Bis (trimethylsilyl) trifluoroacetamide were, respectively, added to the blown sample. The sample was then vortexed for 15 s, reacted under 80°C for 15 min and blew by N2 for dryness. 100 μl of hexane was added before 5 min 25,155 × g centrifugation; the supernatant was extracted for GC/MS analysis.
Agilent 7890A-5975C GC/MS was used for chromatographic analyses. The used chromatographic column was DB-5 (25 m × 200 μm × 0.33 μm) with 1 μl injection volume and a split ratio of 4:1. Programmatic temperature rising with an initial temperature of 70°C, 10°C was raised per minute till 300°C, steady for 4 min. All the data were collected by SIM mode with a scanning range of 40–600 and a solvent delay of 6 min.
Statistical analysis was carried out by using GraphPad Prism version 6.0c for Mac OS, (GraphPad Software, San Diego, California USA). The Chi-square test was used for the sensitivity and specificity analysis, P < 0.05 was considered statistically significant.
| Results|| |
In total, all 140 patients were admitted to undergo TBSA detection in their cerebrospinal fluid, only 27 (27/140, 19.3%) of them were determined as confirmed TBM through gold standard examination. Each patient received at least one of the three gold standard tests, finding that 20, 4, and 5 patients were having positive results in CSF smear (number of tests performed out of 140 patients: 133/140), CSF culture (67/140), and NAAT (96/140), respectively [Table 2]. In which two of the patients with CSF smear-positive also showed a positive result in either CSF culture or NAAT. Among these positive tests, the corresponding numbers of positive TBSA results were shown in the brackets, indicating the patients who could be diagnosed by both gold standard tests and TBSA [Table 2].
|Table 2: Stratification of “Definite” (cerebrospinal fluid smear, cerebrospinal fluid culture, and nucleic acid amplification tests positive) patients into the lancet scoring classification of “Probable,” “Possible,” and “Not”|
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TBSA detection (0.5 nmol/L TBSA in CSF was selected as the cutoff level for a positive result, which is the smallest concentration of TBSA that could be detected by GC/MS under our pretreatment protocol) found that 50 (50/140, 35.7%) and 90 (90/140, 64.3%) patients had positive and negative results, respectively. [Table 3] illustrates the patients' distribution in terms of TBSA and gold standard tests. Among TBSA-positive patients, 20 of them were found with confirmed results of gold standard (i.e., definite TBM), obtaining the sensitivity of TBSA detection of 0.7407 (confidence interval [CI] 95%: 0.5372–0.8889). Specificity, positive predictive value, and negative predictive value were found as 0.7345 (CI 95%: 0.6432–0.8132), 0.4000 (CI 95%: 0.2641–0.5482), and 0.9222 (CI 95%: 0.8463–0.9682), respectively. The Chi-square test showed a statistically significance with P < 0.0001.
|Table 3: Comparison of the gold standard examination and tuberculostearic acid detection for patients' cerebrospinal fluid|
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In terms of the Lancet scoring system, with not considering to group patients into the “Definite” TBM, all patients were classified into “Probable” (n = 35), “Possible” (n = 86) and “Not” (n = 19) TBM groups [Table 4]. Numbers of positive-gold standard as well as positive-TBSA patients were, respectively, stratified to indicate their distribution in each group. Both “Gold Standard” and “TBSA” tests showed a similar pattern of positive rates among groups (Highest: “Probable;” middle: “Possible;” and lowest: “Not”). Although “Possible” TBM showed a moderated positive rate due to including the largest portion of negative-results patients, it comprised the most TBM confirmed and TBSA-positive patients as compare to other groups. Furthermore, patients in the “Not” TBM group could still be found with the positive results of gold standard (three patients) and TBSA detection (four patients). Overall, TBSA showed higher positive rates in the stratified groups and total results than gold standard, indicating its stronger detection capability and higher sensitivity than traditional laboratory diagnostic assays for Mtb existence implication.
|Table 4: Comparison of the stratification of “Definite” and tuberculostearic acid patients|
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| Discussion|| |
To our best knowledge, no new publications could be found for studying the diagnosis of TBM through TBSA detection in cerebrospinal fluid since 2004. However, the methodology of TBSA detection in sputum or other body fluids is still being updated in diagnosis of pulmonary or non-CNS extrapulmonary tuberculosis. Therefore, we deem that the importance of TBSA for tuberculosis diagnosis in CNS should be emphasized to clinicians, especially neurologists, in order to develop an effective and valuable diagnostic system with the help of TBSA.
In this study, we examined 140 patients' CSF specimens to find out the sensitivity and specificity of TBSA for TBM diagnosis. Our results showed discrepancies from the first paper which reported robust results of sensitivity and specificity. Despite the efficiency and accuracy in our study of TBSA detection is lower than previous studies, this assay is still a meaningful method indicating Mtb existence, because TBSA is a cell wall component of Mtb. We observed that TBSA detection always showed a higher sensitivity than gold standard tests, as well as a similar distributive pattern of confirmed TBM results among the Lancet grouping, which gives us clues that TBSA-positive patients might contain the characteristics of the “definite” TBM patients shown [Table 4]. Our results infer that positive TBSA can indicate the existence of Mtb in suspected TBM patients whose direct evidence of TB infection is still unavailable through traditional CSF examinations. Thirty patients in our study were identified as false-positive results [Table 3], but possibly all or any of them were truly infected with Mtb and can be confirmed as TBM in future.
The Lancet consensus scoring system published in 2010 is universally acknowledged in clinical application for TBM suspected patients and is conducive to treatment and prognosis evaluation. However, we noticed that this scoring system could not provide accurate information in classifying unconfirmed TBM patients when gold standard tests fail in diagnosis. According to [Table 4], we found that a lot of TBM confirmed patients were grouped into “Possible” and “Not” TBM rather than “Probable” TBM, that being said, the efficacy of the Lancet scoring system is insufficient for physicians to timely initiate anti-TB therapies for patients. On the other hand, TBSA detection was found to have similarity in distribution with gold standard tests and show higher sensitivity. We believe that the benefit of complementary use of TBSA in diagnosis shines in the way to timely treat patients who suffer from TBM but still awaiting the confirmation. Thus, we recommend that TBSA should be included as one of the evaluation parameters in future scoring systems to compensate for the low-sensitivity gold standard tests. TBSA is believed to have great potentials to provide an accurate diagnosis, entails with earlier treatment for TBM suspected patients.
One of the limitations in our study is that patients did not undergo all of the three gold standard tests due to the variability in clinical decision-making process and concerns of diagnostic efficiency (e.g., CSF culture may require at least 4 weeks). Second, patients with false-positive results of TBSA consist of both true TBM and other bacterial or fungal meningitis patients. Therefore, bacterial and fungal examinations should be conducted in the same cohort to exclude patients who have co-infected with other microorganisms. Last, TBSA was the only analyzed component in our chromatographic analysis. Metabolomics analyses in chromatography study are expected to be carried out for searching multiple biomarkers in CSF for TBM diagnosis in future.
| Conclusions|| |
In summary, we have obtained the sensitivity and specificity of TBSA detection in CSF and revealed the similar distributive pattern of TBSA detection and the gold standard tests in the Lancet grouping. Furthermore, we elaborate the implications of creating a clinical evaluation system including TBSA that might enhance the diagnostic accuracy of TBM. An accurate scoring system, including TBSA detection, needs to be designed with the ability of rapid diagnosis in future research work to further applicate the diagnostic value of TBSA.
This study was approved by the Medica Ethics Committee of Nanfang Hospital, Guangzhou, China (Number of approval letter: NFEC-2018-087).
Financial support and sponsorship
This study was financially supported by Science and Technology Planning Project of Guangdong Province (Grant No. 2017B020247006), China and Natural Science Foundation of Guangdong Province, China (Grant No. 2016A030313579).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]