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Year : 2011  |  Volume : 54  |  Issue : 2  |  Page : 421-422
Early diagnosis of tuberculous meningitis: A comparison of nested polymerase chain reaction and BacT/ALERT

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

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Date of Web Publication27-May-2011

How to cite this article:
Parija SC, Gireesh A R. Early diagnosis of tuberculous meningitis: A comparison of nested polymerase chain reaction and BacT/ALERT. Indian J Pathol Microbiol 2011;54:421-2

How to cite this URL:
Parija SC, Gireesh A R. Early diagnosis of tuberculous meningitis: A comparison of nested polymerase chain reaction and BacT/ALERT. Indian J Pathol Microbiol [serial online] 2011 [cited 2021 Jan 27];54:421-2. Available from: https://www.ijpmonline.org/text.asp?2011/54/2/421/81610


An accurate and early diagnosis of tuberculous meningitis (TBM) is important for its effective management. Conventional techniques lack sensitivity and specificity. [1] While the semi-automated and automated continuously monitored systems have reduced the culture times, the polymerase chain reaction (PCR) is a more rapid and sensitive method for detection of Mycobacterium tuberculosis in clinical samples. A nested PCR has been recommended for cerebrospinal fluid (CSF) where the bacterial load is very low. [2] The present study was carried out to evaluate the performance of a nested PCR using CSF samples obtained from suspected cases of TBM.

About 1.5 ml of CSF was collected under aseptic precautions from each of the 70 clinically suspected cases of TBM and 39 non-TBM controls. Microscopy, culture on Lowenstein Jensen (LJ) medium and BacT/ALERT and nested PCR targeting IS6110 were performed as mentioned earlier [3] and the results were compared. Standard laboratory M. tuberculosis strain H37Rv was used for standardization of DNA extraction and amplification. [4] Two sets of primers were designed for insertion sequence 6110 (IS6110).

Overall, six samples were positive by auramine phenol staining only, of which three samples were positive by both Ziehl Neelsen (ZN) and auramine phenol staining methods and by culture on the LJ medium and by the nested PCR. The rest were negative by culture on the LJ medium. Five samples were positive for M. tuberculosis by the BacT/ALERT system with a mean detection time for M. tuberculosis of 13.2 days. All the control samples were negative by all of the above methods [Figure 1]. The ZN and auramine phenol smear examination had a sensitivity of 2.85% and a specificity of 100%. Growth of M. tuberculosis was detected in 3/70 (4.2%) of the CSF samples with a sensitivity of 2.85% and specificity of 100%. BacT/ALERT culture showed a sensitivity of 7.14% and a specificity of 77.7%. No other mycobacterial species were isolated. In comparison, nested PCR was found to have a much higher sensitivity of 40% and a specificity of 100%. In this study, only 1.5 ml was collected; therefore, the sensitivity of smear microscopy was lower as compared to earlier citations. [5] PCR showed the highest sensitivity as compared to the other tests and detected M. tuberculosis in less than 2 days, similar to the earlier studies. In this study, the first set PCR showed lower sensitivity (7.1%) in detecting the TBM cases. With the use of nested PCR, we were able to detect M. tuberculosis in 37.31% smear negative samples.
Figure 1: Gel image showing 219 bp target amplicon from CSF; M = DNA ladder, PC = positive control, NC = negative control, 1-4 are positive cases

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In conclusion, molecular diagnosis of TBM by nested PCR has a great potential to improve the ability to diagnose TBM. Nested PCR is highly specific and sensitive than simplex PCR for the diagnosis of TBM where the concentration of tubercle bacilli is low. Since the diagnosis of TBM by using conventional methods alone is difficult, the nested PCR is a useful adjunct for the rapid and specific diagnosis of TBM. Early detection of the disease ensures early treatment of the patients which will finally go a long way in reducing the burden of tuberculosis.

   References Top

1.Mathai A, Radhakrishnan VV, George SM, Sarada C. A newer approach for the laboratory diagnosis of tuberculous meningitis. Diagn Microbiol Infect Dis 2001;39:225-8.  Back to cited text no. 1
2.Yuen KY, Yam WC, Wong LP, Seto WH. Comparison of two automated DNA amplification systems with a manual one-tube nested PCR assay for diagnosis of pulmonary tuberculosis. J Clin Microbiol 1997;35:1385-9.  Back to cited text no. 2
3.Koneman EW. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 6 th ed. Philadelphia: Lippincott Williams and Wilkins, Wolters Kluwer Company; 2006.  Back to cited text no. 3
4.Miyazaki Y, Koga H, Kohno S, Kaku M. Nested polymerase chain reaction for detection of mycobacterium tuberculosis in clinical samples. J Clin Microbiol 1993;31:2228-32.  Back to cited text no. 4
5.Bhargava S, Gupta AK, Tandon PN. Tuberculous meningitis-A CT study. Br J Radiol 1982;55:189-96.  Back to cited text no. 5

Correspondence Address:
Subhash Chandra Parija
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.81610

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