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Validation of Mycobacterium tuberculosis real-time polymerase chain reaction for diagnosis of tuberculous meningitis using cerebrospinal fluid samples: a pilot study

  • Sérgio M. de Almeida EMAIL logo , Conrado M. Borges , Lucas B. Santana , Gilberto Golin , Lísia Correa , Gislene B. Kussen and Keite Nogueira
Published/Copyright: September 29, 2018
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 57 Issue 4

Abstract

Background

Timely diagnosis of tuberculous meningitis (TBM) remains challenging. Molecular diagnostic tools are necessary, particularly in low- and middle-income countries. There is no approved commercial polymerase chain reaction (PCR) assay that can be used to detect Mycobacterium tuberculosis in non-respiratory samples, such as the cerebrospinal fluid (CSF). We aimed to validate the threshold cycle (Ct) cut-off points; calculate the operational characteristics of real-time PCR for detection of M. tuberculosis (MTb qPCR) in the CSF; and the inhibitory affect of CSF red blood cells (RBC) and total proteins on MTb qPCR.

Methods

A total of 334 consecutive participants were enrolled. Based on clinical, laboratory and imaging data, cases of suspected TBM were categorized as definite, probable, possible or not TBM cases. Receiver operating characteristic curve analysis was used to select the best discriminating Ct value.

Results

For TBM cases categorized as definite or probable (n=21), the Ct validated for CSF (≤39.5) improved the diagnostic performance of MTb qPCR on CSF samples. The sensitivity was 29%, specificity was 95%, positive predictive value was 26%, negative predictive value was 95%, efficiency was 90% and positive likelihood was 5.3. The CSF RBC and total protein did not affect the positivity of the MTb qPCR.

Conclusions

These data support the validation of a highly specific but low sensitive MTb qPCR assay for the TBM diagnosis using CSF samples. MTb qPCR contributes significantly to the diagnosis, mainly when associated with conventional microbiology tests and clinical algorithms.

Keywords: central nervous system; diagnosis; inhibition; meningeal tuberculosis; polymerase chain reaction; tuberculosis; tuberculous meningitis (TBM)
Corresponding author: Sérgio M. de Almeida, MD, PhD Hospital de Clínicas-UFPR, Seção de Virologia, Setor Análises Clínicas, Rua Padre Camargo, 280, Curitiba, PR, 80060-240, Brazil

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Wilder-Smith EP. Mycobacterium tuberculosis. In: Lisak RP, Truong DD, Carroll WM, Bhidayasiri R, editors. International neurology, a clinical approach. Oxford, UK: Blackwell Publishing, 2009:258–61.Search in Google Scholar

2. World Health Organization. 2009. Resolution WHA62.15. Prevention and control of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. In: Sixty-second World Health Assembly, Geneva, 18–22 May 2009, Resolutions and decisions; annexes. Geneva, Switzerland: World Health Organization, 2009 (WHA62/2009/REC/1):25–9.Search in Google Scholar

3. Roca B, Tornador N, Tornador E. Presentation and outcome of tuberculous meningitis in adults in the province of Castellon, Spain: a retrospective study. Epidemiol Infect 2008;136:1455–62.10.1017/S0950268807000258Search in Google Scholar

4. Pai M, Flores LL, Pai N, Hubbard A, Riley LW, Colford JM Jr. Diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: a systematic review and meta-analysis. Lancet Infect Dis 2003;3:633–43.10.1016/S1473-3099(03)00772-2Search in Google Scholar

5. Fishman RA. Cerebrospinal fluid in diseases of the nervous system, 2nd ed. Philadelphia, PA: WB Saunders, 1992.Search in Google Scholar

6. Thakur R, Goyal R, Sarma S. Laboratory diagnosis of tuberculous meningitis – is there a scope for further improvement. J Lab Physicians 2010;2:21–4.10.4103/0974-2727.66705Search in Google Scholar

7. Philip N, William T, John DV. Diagnosis of tuberculous meningitis: challenges and promises. Malays J Pathol 2015;37:1–9.Search in Google Scholar

8. Sun Q, Sha W, Xiao HP, Tian Q, Zhu H. Evaluation of cerebrospinal fluid adenosine deaminase activity for the differential diagnosis of tuberculous and nontuberculous meningitis. Am J Med Sci 2012;344:116–21.10.1097/MAJ.0b013e318238fee3Search in Google Scholar

9. Katoch VM. Newer diagnostic techniques for tuberculosis. Indian J Med Res 2004;120:418–28.Search in Google Scholar

10. Chaidir L, Ganiem AR, vander Zanden A, Muhsinin S, Kusumaningrum T, Kusumadewi I, et al. Comparison of real time IS6110-PCR, microscopy, and culture for diagnosis of tuberculous meningitis in a cohort of adult patients in Indonesia. PLoS One 2012;7:e52001.10.1371/journal.pone.0052001Search in Google Scholar

11. Cho WH, Won EJ, Choi HJ, Kee SJ, Shin JH, Ryang DW, et al. Comparison of AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR for detection of Mycobacterium tuberculosis complex in routine clinical practice. Ann Lab Med 2015;35:356–61.10.3343/alm.2015.35.3.356Search in Google Scholar

12. Tortoli E, Urbano P, Marcelli F, Simonetta MT, Cirillo MD. Is real-time PCR better than conventional PCR for Mycobacterium tuberculosis complex detection in clinical samples? J Clin Microbiol 2012;50:2810–3.10.1128/JCM.01412-12Search in Google Scholar

13. Dayal R, Senthilkumar P, Katoch VM, Chauhan DS, Yadav NK. Diagnostic value of real-time PCR for neurotuberculosis. Indian Pediatr 2010;47:631–2.Search in Google Scholar

14. 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;10:803–12.10.1016/S1473-3099(10)70138-9Search in Google Scholar

15. Ratnamohan VM, Cunningham AL, Rawlinson WD. Removal of inhibitors of CSF-PCR to improve diagnosis of herpesviral encephalitis. J Virol Methods 1998;72:59–65.10.1016/S0166-0934(98)00020-2Search in Google Scholar

16. Almeida SM, Raboni SM, Noqueira MB, Vidal LR. Red blood cells in cerebrospinal fluid as possible inhibitory factor for enterovirus RT-PCR. Arg Neuropsiquiatr 2016;74:810–5.10.1590/0004-282X20160119Search in Google Scholar PubMed

17. Lee CK, Chai CN, Capinpin SM, Ang A, Ng SY, Lee PL, et al. Evaluation of the Luminex ARIES HSV 1 and 2 Assay and comparison with the FTD Neuro 9 and In-house Real-Time PCR Assays for detecting Herpes Simplex Viruses. Ann Lab Med 2018;38:440–5.10.3343/alm.2018.38.5.440Search in Google Scholar PubMed PubMed Central

18. White S, Schultz T, Enuameh YA. Synthesizing evidence of diagnostic accuracy. Philadelphia, PA: Lippincott Williams and Wilkins, 2011.Search in Google Scholar

19. Kent PT. Public health mycobacteriology: a guide for the level III laboratory. Atlanta, GA: Centers for Disease Control, 1985.Search in Google Scholar

20. Akobeng AK. Understanding diagnostic tests 2: likelihood ratios, pre- and post-test probabilities and their use in clinical practice. Acta Pædiatr 2006;96:487–91.10.1111/j.1651-2227.2006.00179.xSearch in Google Scholar PubMed

21. Akobeng AK. Understanding diagnostic tests 3: receiver operating characteristic curves. Acta Pædiatr 2007;96:644–7.10.1111/j.1651-2227.2006.00178.xSearch in Google Scholar PubMed

22. Galen RS, Gambino SR. Beyond normality, the predictive value and efficiency of medical diagnoses. New York, NY: Wiley & Sons, 1975:237.Search in Google Scholar

23. Mitchell AJ. The clinical significance of subjective memory complaints in the diagnosis of mild cognitive impairment and dementia: a meta-analysis. Int J Geriatr Psychiatry 2008;23:1191–202.10.1002/gps.2053Search in Google Scholar PubMed

24. Mitchell AJ. Sensitivity x PPV is a recognized test called the clinical utility index (CUI+). Eur J Epidemiol 2011;26:251–2.10.1007/s10654-011-9561-xSearch in Google Scholar PubMed

25. McGee S. Simplifying likelihood ratios. J Gen Intern Med 2002;17:647–50.10.1046/j.1525-1497.2002.10750.xSearch in Google Scholar PubMed PubMed Central

26. Sackett DL, Haynes RB. The architecture of diagnostic research. Br Med J 2002;324:539–41.10.1136/bmj.324.7336.539Search in Google Scholar PubMed PubMed Central

27. Fagan TJ. Letter- nomogram for Bayes theorem. N Engl J Med 1975;293:257.10.1056/NEJM197507312930513Search in Google Scholar PubMed

28. Burd EM. Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev 2010;23: 550–76.10.1128/CMR.00074-09Search in Google Scholar PubMed PubMed Central

29. CLSI. Molecular diagnostic methods for infectious diseases, 3rd ed., CLSI reports MM03. Wayne, PA: Clinical and Laboratory standards Institute, 2015.Search in Google Scholar

30. Boyko EJ. Ruling out or ruling in disease with the most sensitive or specific diagnostic test: short cut or wrong turn? Med Decis Making 1994;14:175–9.10.1177/0272989X9401400210Search in Google Scholar PubMed

31. Babafemi EO, Cherian BP, Banting L, Mills GA, Ngianga II K. Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis. Syst Rev 2017;6:215.10.1186/s13643-017-0608-2Search in Google Scholar

32. Bahr NC, Marais S, Caws M, van Crevel R, Wilkinson RJ, Tyagi JS, et al. GeneXpert MTB/Rif to diagnose tuberculous meningitis: perhaps the first test but not the last. Clin Infect Dis 2016;62:1133–5.10.1093/cid/ciw083Search in Google Scholar

33. Bahr NC, Nuwagira E, Evans EE, Cresswell FV, Bystrom PV, Byamukama A, et al. Diagnostic accuracy of Xpert MTB/RIF Ultra for tuberculous meningitis in HIV-infected adults: a prospective cohort study. Lancet Infect Dis 2018;18:68–75.10.1016/S1473-3099(17)30474-7Search in Google Scholar

34. Chiang SS, Khan FA, Milstein MB, Tolman AW, Benedetti A, Starke JR, et al. Treatment outcomes of childhood tuberculous meningitis: a systematic review and meta-analysis. Lancet Infect Dis 2014;14:947–57.10.1016/S1473-3099(14)70852-7Search in Google Scholar

35. Kumar K, Giribhattanavar P, Chandrashekar N, Patil S. Correlation of clinical, laboratory and drug susceptibility profiles in 176 patients with culture positive TBM in a tertiary Neurocare centre. Diagn Microbiol Infect Dis 2016;86:372–6.10.1016/j.diagmicrobio.2016.09.018Search in Google Scholar PubMed

Received: 2018-05-16
Accepted: 2018-08-17
Published Online: 2018-09-29
Published in Print: 2019-03-26

©2019 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2018-0524
Keywords for this article
central nervous system; diagnosis; inhibition; meningeal tuberculosis; polymerase chain reaction; tuberculosis; tuberculous meningitis (TBM)

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Can Grail find the trail to early cancer detection?
  4. Reviews
  5. Aberrant glycosylation and cancer biomarker discovery: a promising and thorny journey
  6. Application of NMR metabolomics to search for human disease biomarkers in blood
  7. Mini Review
  8. Host-response biomarkers for the diagnosis of bacterial respiratory tract infections
  9. Opinion Paper
  10. Diabetes alert dogs: a narrative critical overview
  11. EFLM Paper
  12. Documenting metrological traceability as intended by ISO 15189:2012: A consensus statement about the practice of the implementation and auditing of this norm element
  13. General Clinical Chemistry and Laboratory Medicine
  14. Commutability of external quality assessment materials for serum sodium and potassium measurements
  15. Standardization of measurement procedures for serum uric acid: 8-year experience from Category 1 EQA program results in China
  16. Next-generation rapid serum tube technology using prothrombin activator coagulant: fast, high-quality serum from normal samples
  17. Cannabinoids determination in bronchoalveolar lavages of cannabis smokers with lung disease
  18. A new method for the determination of ammonium in the vitreous humour based on capillary electrophoresis and its preliminary application in thanatochemistry
  19. Reference Values and Biological Variations
  20. Defining reference intervals for a serum growth differentiation factor-15 (GDF-15) assay in a Caucasian population and its potential utility in diabetic kidney disease (DKD)
  21. Cancer Diagnostics
  22. Preanalytical stability of [-2]proPSA in whole blood stored at room temperature before separation of serum and plasma: implications to Phi determination
  23. Cardiovascular Diseases
  24. A de novo mutation of SMYD1 (p.F272L) is responsible for hypertrophic cardiomyopathy in a Chinese patient
  25. Infectious Diseases
  26. Analyzing the capability of PSP, PCT and sCD25 to support the diagnosis of infection in cancer patients with febrile neutropenia
  27. Mid-regional pro-adrenomedullin predicts poor outcome in non-selected patients admitted to an intensive care unit
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  29. Letters to the Editor
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