Immunological diagnosis as an adjunctive tool for an early diagnosis of tuberculous meningitis of an immune competent child in a low tuberculosis endemic country: a case report
© The Author(s) 2017
Received: 18 August 2016
Accepted: 1 March 2017
Published: 13 March 2017
Pediatric tuberculous meningitis is a highly morbid, often fatal disease. Its prompt diagnosis and treatment saves lives, in fact delays in the initiation of therapy have been associated with high mortality rates.
This is a case of an Italian child who was diagnosed with tuberculous meningitis after a history of a month of headache, fatigue and weight loss. Cerebrospinal fluid analysis revealed a lymphocytic pleocytosis with predominance and decreased glucose concentration. Microscopy and conventional diagnostic tests to identify Mycobacterium tuberculosis were negative, while a non classical method based on intracellular cytokine flow cytometry response of CD4 cells in cerebral spinal fluid helped us to address the diagnosis, that was subsequently confirmed by a nested polymerase chain reaction amplifying a 123 base pair fragment of the M. tuberculosis DNA.
We diagnosed tuberculous meningitis at an early stage through an innovative immunological approach, supported by a nested polymerase chain reaction for detection of M. tuberculosis DNA. An early diagnosis is required in order to promptly initiate a therapy and to increase the patient’s survival.
KeywordsTuberculous meningitis Children Hydrocephalus Immunological diagnosis ICCFC
Tuberculous meningitis (TBM) represents roughly 1% of all cases of TB and it is associated with high mortality and residual neurologic sequelae, even with adequate treatment. In endemic countries the highest incidence of TBM is reported in children aged 2–4 years . Early diagnosis is notoriously difficult and often delayed and it has long been recognized as the single most important factor determining outcome . In fact the microbiological diagnosis of TBM is difficult due to the paucibacillary nature of the cerebrospinal fluid (CSF) in which the microscopy for acid-fast bacilli (AFB) and culture for Mycobacterium (M.) tuberculosis have a low sensitivity [1, 3, 4]. Also commercial nucleic acid amplification tests (NAATs) showed a low sensitivity and specificity compared to culture for the diagnosis of TBM . An additional test is the Adenosine deaminase (ADA) measurements in CSF , even if the specificity is low and the cut-off level has not been determined .
We present a case study of an Italian child with TBM who was hospitalized in the Pediatric Infectious diseases department of Umberto I° Hospital in Rome, and in whom the early diagnosis, based on immunological flow cytometry test and molecular assay, allowed us to start an early treatment, fundamental requirement to increase patient’s survival.
In order to improve the diagnosis of TB, we performed an alternative immunological method based on multifunctional T cells, which has been suggested in recent years as a new tool for the discrimination between active TB and latent TB infection (LTBI) . In a previous study  we used an intracellular cytokine flow cytometry (ICCFC) protocol to asses mono-functional and multi-functional Mtb-specific CD4+ in peripheral blood and we proposed an immune-based approach, which could improve the identification at single time point of subjects with no TB infection or patients having active or latent TB. A receiver operating characteristic (ROC) analysis was performed to calculate optimal cut-off values for both activated CD4+ T cells and polyfunctional CD4+ T cells in order to discriminate infected (active TB and LTBI) from uninfected patients and active from latent stage. We performed a ROC analysis, and a cut-off >0.45% for activated CD4+ T cells was found as the value allowing the best combination of sensitivity (94.44%, 95% CI 72.2–99.8%) and specificity (100%, 95% CI 69.15–100%; AUC 0.9722; 95% CI 0.9141–1.030%, P < 0.0001) to differentiate Mtb-infected patients (active TB and LTBI) from healthy controls . A further cut-off <0.182% for polyfunctional CD4+ T cells allowed the best combination of sensitivity (77.78%) and specificity (70%) to differentiate between active TB and LTBI subjects.
Although this study involved pulmonary TB, we tried to apply our algorithm to TB meningitis. The same method to stain peripheral blood was used to stain CSF. CSF (0.5 mL) was added to the 3 tubes of QFT-IT containing respectively, saline solution (negative control), phytohaemagglutinin (positive control), and TB antigens (ESAT-6, CFP-10, and TB 7.7). A costimulation with 5 μL/mL anti-CD28 plus anti-CD49d (BD Bioscience, Pharmingen, Italy) and 10 μg/mL BrefeldinA (Sigma-Aldrich) was added in all tubes . After 18 h of incubation, the cell surface staining was performed with the markers anti-CD45-VioBlue and anti-CD4 PE-Vio770 (Miltenyi Biotec, Germany), then the cells were lysed (BD Bioscience Lyse solution) and permeabilized (BD Bioscience Perm solution) and the intracellularly stained with anti-IFN-γ FITC, anti-TNF-α APC and anti-IL-2 PE (Miltenyi Biotec). Eventually cells were acquired with a MACSQuant Analyzer flow cytometer (Miltenyi Biotec) and analysed with FlowJo Software version 10, that allowed us to perform a “combination gates” analysis. Seven different population cells were detected in CD4+ cell gate on the basis of IFN-γ, IL-2, and TNF-α produced by CD4+ (Additional file 1), as previously described .
Moreover we carried out on the CSF extracted from the LP in the first day of hospitalization, the l ADA level which amounted to 18.31 UI/L, higher than the cut-off 10 .
Two days later, due to persistent clinical symptoms and uncertainty in the tests of coordination, a second brain MRI was performed with a report of hypertensive hydrocephalus and a diffuse leptomeningeal enhancement of the basal cistern, in particular of the interpeduncular cistern, attributable to infectious-inflammatory alterations. The brain MRI is shown in Fig. 1b, c. An encephalic drainage was inserted urgently.
Based on the clinical, radiological findings and CSF results (CD4+ polyfunctional response, ADA level, pleocytosis, hyperproteinorrachia and hypoglicorrachia) TBM was considered on admission day 3 and therapy was started with antituberculous drugs with isoniazid (H) 300 mg/day plus ethambutol (E) 500 mg/day plus rifampicin (R) 400 mg/day and pyrazinamide (Z) 1000 mg/day, as well as intravenous steroids (dexamethasone 4 mg every 8 h).
Serial CSF data from lumbar puncture and ventricular drain
Day 1 CSF-LP
Day 9 CSF-LP
Day 14 CSF- LP
Day 18 CSF- LP
Day 18 CSF-VD
Day 21 CSF-LP
Day 21 CSF-VD
Day 27 CSF-LP
Day 27 CSF-VD
Day 40 CSF-LP
Day 52 CSF-LP
Protein level mg/dL
Lactic acid mg/dL
Direct gram stain
PCR M. tuberculosis
Culture M. tuberculosis
Bacterioscopic exam for AFB
Renal and liver function, bilirubin and uric acid levels were monitored regularly. Serial CSF values are shown in Table 1. On day 9 a “in house” nested PCR amplifying a 123 base pair fragment of the M. tuberculosis DNA was performed using CSF with a positive result, while the The GeneXpert MTB/RIF assay was negative.
The family screening was performed and only the father was found TST positive with normal chest radiography and chest CT scan.
Once the patient’s condition stabilized, the HERZ regimen was continued and dexamethasone was gradually decreased. However, on admission day 27, because of an acute onset of aphasia with right hemiplegia, an emergency brain MRI was performed (Fig. 1d). The findings were compatible with tuberculous cerebral vasculitis (TVC), resulting in a diagnosis of ischemia with two focal areas of signal restriction in correspondence of the left caudate nucleus and of the posterior arm of the left internal capsule. We increased the corticosteroid dosage and treatment with enoxaparin and aspirin was added.
On admission day 74, there were no further episodes of fever, the neurological examination was normal with total disappearance of hemiplegia, the facial asymmetry and the double vision, thanks to neurological rehabilitation. The patient was discharged in good condition without significant neurological sequelae besides the presence of a slight disorder of attention and concentration and with recommendation to continue the treatment with HERZ regimen and aspirin. She was followed up at the outpatient clinic and a follow-up MRI of brain is shown on the Fig. 1d. Child is on regular follow-up.
Discussion and conclusions
We report a case of TBM complicated with hydrocephalus and cerebral vasculitis tuberculosis (TVC), which benefited from early diagnosis, based on unconventional methods, and an early TB treatment increasing the chance of a favourable outcome.
TBM remains the most lethal form of M. tuberculosis and a recent large childhood study documented neurological disability in about 75% of survivors, despite an exceptionally low mortality rate of only 13% and a treatment with antituberculosis chemotherapy .
Early diagnosis and treatment of TBM is the single most important factor determining outcome [13–15]. Although prompt and rapid identification of TBM is crucial for a successful disease management, in most cases, the diagnosis is significantly delayed with a consequent delay in the initiation of therapy, which is often attributable to the use of slow or relatively insensitive conventional diagnostic tests .
The diagnosis of TBM is difficult because of unspecific symptoms and signs.
There are some evidences that a combination of clinical and simple laboratory data might help in the diagnosis . Certain clinical characteristics such as longer duration of symptoms (>6 days), moderate CSF pleocytosis, and the presence of focal deficits increase the probability of TBM [17–19]. Characteristic CSF findings of TBM include lymphocytic-predominant pleocytosis, a total white cell count of 100–500 cells/μL, elevated protein levels (typically between 100 and 500 mg/dL) and low glucose, usually less than 45 mg/dL or CSF: plasma ratio <0.5 . Marais et al.  have created a score based on clinical, CSF data and cerebral imaging criteria plus evidence of TB elsewhere. According to these authors, probable TBM is defined by a score between 10 and 12, whereas possible TBM is defined by a score higher than 6. At the admission to our ward the patient obtained a Marais score of 6 (4 scores for the CSF findings and 2 scores for clinical criteria) that was insufficient to speculate a TBM diagnosis.
In our case report neither the microscopy, to detect acid-fast bacilli in the CSF, nor culture resulted positive since TBM is a paucibacillary form of tuberculosis so they were unhelpful in making diagnosis. The likelihood of seeing or culturing M. tuberculosis from the CSF is dependent upon meticulous microscopy and culture of a large volume (>5 mL) of CSF  that is difficult in most cases, especially in children. In fact microscopy, although rapid and inexpensive, has very low sensitivity (10–20%), while culture is too slow and insensitive (<50%) to aid clinical decision-making .
In the last decades nucleic acid-based amplification (NAA) tests have emerged as potentially important tools for diagnosing TBM, unfortunately they have a high specificity (97–99%) but low sensitivity (46–66%) . In our patient the first molecular test used (GeneXpert MTB/RIF assay, Cepheid) was negative. Improving the conditions of the CSF sample pre-treatment prior to DNA extraction and increasing the sensitivity of molecular test using a nested PCR , it was possible to confirm the suspect of TBM in our patient.
To increase the chance to detect TB infection we used whole blood QFT-G-IT (an Interferon Gamma Release Assay, IGRA) to detect the response to stimuli with specific TB antigens. Our child obtained an IGRA on whole blood indeterminate, in accordance with Vidhate and colleagues  who found that whole blood QFT-G-IT had low sensitivity and specificity in diagnosing TBM and CSF QFT-G-IT was useful neither in diagnosis nor in predicting the outcome, due to indeterminate results.
Furthermore we performed an innovative assay to detect the CD4 intracellular response in whole blood and CSF based on the premise that mononuclear cells localised to infected sites produce more cytokines than peripheral blood mononuclear cells (PBMC) , which has also been demonstrated in other tissues [30, 31]. Intracellular response in peripheral blood showed firstly a TB infection, revealed by neither TST nor Quantiferon. Moreover the polyfunctional CD4 profile suggested a pattern associated to active TB, according to the previously proposed algorithm . The same test performed in CSF confirmed a M. tuberculosis infection with a high local immune specificity. In CSF the increased proportion of CD4 cells producing cytokines (IL-2, INF-γ, TNF-α) could reflect the high number of mononuclear cells localised to infected sites, typical of TBM. A similar phenomenon was described in pleural  and alveolar fluid  in thoracic TB.
In our study the use of non conventional methods to detect TBM, such as intracellular staining of peripheral blood and of CSF followed by the detection of ADA activity and a in house nested PCR, helped us in the early diagnosis and treatment of TBM, despite the consecutive negativity of classical diagnostic tests. A prospective study with a large number of children is needed to confirm this isolate observation.
cerebral spinal fluid
intracellular cytokine flow cytometry
nucleic acid-based amplification
SV and ML designed the study. SV, EC and ML drafted the manuscript. CA, EC, MRC, CDB and VV contributed to the treatment of the clinical case. CrM and DC contributed to the neurosurgical management. ClM and FM carried out the immunological assays. MGP and FM contributed to the microbiological assays. SV and ML reviewed and edited the manuscript. CMM reviewed and supervised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
Written informed consent was obtained from the parent of the patient for publication of this Case Report and any accompanying images.
Ethics approval and consent to participate
Ethical approval to report this case was not required.
This study was supported by in part by SIMIT Grant 2015.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- van Well GT, Paes BF, Terwee CB, Springer P, Roord JJ, Donald PR, et al. Twenty years of pediatric tuberculous meningitis: a retrospective cohort study in the western cape of South Africa. Pediatrics. 2009;123:e1–8.View ArticlePubMedGoogle Scholar
- Lincoln EM, Sordillo SUR, Davies PA. Tuberculous meningitis in children. J Pediatr. 1960;57:807–23.View ArticlePubMedGoogle Scholar
- Jönsson B, Ridell M. The Cobas Amplicor MTB Test for detection of Mycobacterium tuberculosis complex from respiratory and non-respiratory clinical specimens. Scand J Infect Dis. 2003;35:372–7.View ArticlePubMedGoogle Scholar
- Hosoglu S, Geyik MF, Balik I, Aygen B, Erol S, Aygencel TG, et al. Predictors of outcome in patients with tuberculous meningitis. Int J Tuberc Lung Dis. 2002;6:64–70.PubMedGoogle Scholar
- Solomons RS, van Elsland SL, Visser DH, Hoek KG, Marais BJ, Schoeman JF, et al. Commercial nucleic acid amplification tests in tuberculous meningitis—a meta-analysis. Diagn Microbiol Infect Dis. 2014;78:398–403.View ArticlePubMedGoogle Scholar
- Fox IH, Kelly WN. The role of adenosine deaminase and 2′deoxyadenosine in mammalian cells. Ann Rev Biochem. 1978;47:655–86.View ArticlePubMedGoogle Scholar
- CDC-Taiwan. Taiwan guideline on TB diagnosis and treatment. In: CDC-Taiwan. 4nd ed. Taipei: Taiwan Centers for Disease Control; 2012.
- Link H. Contribution of CSF studies to diagnosis of multiple sclerosis. Ital J Neurol Sci. 1987;6:57–69.PubMedGoogle Scholar
- Harari A, Rozot V, Bellutti Enders F, Perreau M, Stalder JM, Nicod LP, et al. Dominant TNF-α + Mycobacterium tuberculosis-specific CD4+ T cell responses discriminate between latent infection and active disease. Nat Med. 2011;17:372–7.View ArticlePubMedGoogle Scholar
- Lichtner M, Mascia C, Sauzullo I, Mengoni F, Vita S, Marocco R, et al. Multifunctional analysis of CD4+ T-cell response as immune-based model for tuberculosis detection. J Immunol Res. 2015;2015:217287.View ArticlePubMedPubMed CentralGoogle Scholar
- González-Duarte A, Ponce de León A, Osornio JS. Importance of differentiating Mycobaterium bovis in tuberculous meningitis. Neurol Int. 2011;3(3):e9.View ArticlePubMedPubMed CentralGoogle Scholar
- Tuon FF, Higashino HR, Lopes MI, Litvoc MN, Atomiya AN, Antonangelo L, et al. Adenosine deaminase and tuberculous meningitis—a systematic review with meta-analysis. Scand J Infect Dis. 2010;42(3):198–207.View ArticlePubMedGoogle Scholar
- Fallon RJ, Kennedy DH. Treatment and prognosis in tuberculous meningitis. J Infect. 1981;3:39–44.View ArticlePubMedGoogle Scholar
- Misra UK, Kalita J, Srivastava M, Mandal SK. Prognosis of tuberculous meningitis: a multivariate analysis. J Neurol Sci. 1996;137:57–61.View ArticlePubMedGoogle Scholar
- Kalita J, Misra UK. Outcome of tuberculous meningitis at 6 and 12 months: a multiple regression analysis. Int J Tuberc Lung Dis. 1999;3:261–5.PubMedGoogle Scholar
- Thwaites G, Fisher M, Hemingway C, Scott G, Solomon T, Innes J. British Infection Society guidelines for the diagnosis and treatment of tuberculosis of the central nerve system in adults and children. J Infect. 2009;59:167–87.View ArticlePubMedGoogle Scholar
- Thwaites GE, Chau TT, Stepniewska K, Phu NH, Chuong LV, Sinh DX, et al. Diagnosis of adult tuberculous meningitis by use of clinical and laboratory features. Lancet. 2002;360:1287–92.View ArticlePubMedGoogle Scholar
- Kumar R, Kohl N. A diagnostic rule for tuberculous meningitis. Arch Dis Child. 1999;81:221–4.View ArticlePubMedPubMed CentralGoogle Scholar
- Marais S, Thwaites G, Schoeman JF, Török ME, Misra UK, Prasad K, et al. Tuberculous meningitis: a uniform case definition for use in clinical research. Lancet Infect Dis. 2010;10:803–12.View ArticlePubMedGoogle Scholar
- Marx GE, Chan ED. Tuberculous meningitis: diagnosis and treatment overview. Tuberc Res Treat. 2011;2011:798764.PubMedPubMed CentralGoogle Scholar
- Thwaites GE, Chau TT, Farrar JJ. Improving the bacteriological diagnosis of tuberculous meningitis. J Clin Microbiol. 2004;42:378–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Pai M, Flores LL, Pai N, Hubbard A, Riley LM, Colford JM. Diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: a systematic review and meta-analysis. Lancet infect Dis. 2003;3:633–43.View ArticlePubMedGoogle Scholar
- Scarpellini P, Racca S, Cinque P, Delfanti F, Gianotti N, Terreni MR, et al. Nested polymerase chain reaction for diagnosis and monitoring treatment response in AIDS patients with tuberculous meningitis. AIDS. 1995;9(8):895–900.View ArticlePubMedGoogle Scholar
- Vidhate MR, Singh MK, Garg RK, Verma R, Shukla R, Goel MM, et al. Diagnostic and prognostic value of Mycobacterium tuberculosis complex specific interferon gamma release assay in patients with tuberculous meningitis. J Infect. 2011;62(5):400–3.View ArticlePubMedGoogle Scholar
- Segura RM, Pascual C, Ocaña I, Martínez-Vázquez JM, Ribera E, Ruiz I, et al. Adenosine deaminase in body fluids: a useful diagnostic tool in tuberculosis. Clin Biochem. 1989;22:141–8.View ArticlePubMedGoogle Scholar
- Baro M, Acevedo L, Lagos ME. Usefulness of adenosine deaminase determination in cerebrospinal fluid for the diagnosis of meningeal tuberculosis; 4 years experience at a public hospital. Rev Med Chile. 1996;124:319–26.PubMedGoogle Scholar
- Pettersson T, Klockars M, Weber TH. Diagnostic value of cerebrospinal fluid adenosine deaminase determination. Scand J Infect Dis. 1991;23:97–100.View ArticlePubMedGoogle Scholar
- Guisti G, Galanti B. Colorimetric method. In: Bergmeyer H, editor. Methods of enzymatic analysis. 3rd ed. Weinheim: Verlag Chemic; 1974. p. 315–23.Google Scholar
- Kim SH, Chu K, Choi SJ, Song KH, Kim HB, Kim NJ, et al. Diagnosis of central nervous system tuberculosis by T-cell-based assays on peripheral blood and cerebrospinal fluid mononuclear cells. Clin Vaccine Immunol. 2008;15:1356–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Losi M, Bossink A, Codecasa L, Jafari C, Ernst M, Thijsen S, et al. Use of a T-cell interferon-c release assay for the diagnosis of tuberculous pleurisy. Eur Respir J. 2007;30:1173–9.View ArticlePubMedGoogle Scholar
- Jafari C, Ernst M, Kalsdorf B, Greinert U, Diel R, Kirsten D, et al. Rapid diagnosis of smear-negative tuberculosis by bronchoalveolar lavage enzyme-linked immunospot. Am J Respir Crit Care Med. 2006;174:1048–54.View ArticlePubMedGoogle Scholar