A Study on Comparison and Evaluation of Sensitivity and Specificity of Different Staining Methods with Conventional Polymerase Chain Reaction among Meningitis Cases - A Prospective Cross Sectional Study (2025)

Accurate diagnosis of tuberculosis meningitis using polymerase chain reaction

International journal of leprosy and other mycobacterial diseases : official organ of the International Leprosy Association, 1994

Utility of polymerase chain reaction in diagnosis of tuberculosis in our setup: a ten years experience

Journal of the College of …, 2012

Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis

Journal of Clinical Microbiology

The complexity, expense, and susceptibility to contamination of the polymerase chain reaction (PCR) are all issues which need to be overcome if PCR is to be used outside of research laboratories. We addressed these problems with respect to the diagnosis of tuberculosis. First, we simplified the procedure for extracting Mycobacterium tuberculosis DNA from sputum samples. Two methods of sample preparation were compared: the chaotrope-silica method and a novel, more simple chloroform method. Second, we developed a colorimetric method for product detection. This method was as sensitive and specific as agarose gel electrophoresis for detection of PCR product. By using a one-tube nested protocol, 5 to 50 genome equivalents of M. tuberculosis DNA were detected. The simplified colorimetric PCR was compared with microscopy and culture for detection of M. tuberculosis in clinical specimens of sputum. A total of 171 sputum samples were investigated from 108 patients, 12 of whom were subsequently found to have tuberculosis by culture and/or microscopy. PCR of samples prepared by the chaotrope-silica method had a sensitivity of 75% and a specificity of 100%/ whereas PCR of samples prepared by the chloroform method had a sensitivity of 92% and a specificity of 99%o when compared with the sensitivities and specificities of the combined classical microbiological methods for the diagnosis of tuberculosis. The simplified colorimetric PCR in combination with the chloroform sample preparation method was at least as sensitive as microscopy but had a greater specificity because samples with atypical mycobacteria were not detected by PCR. The sensitivity of the method for detection of smear-negative and extrapulmonary tuberculosis remains to be investigated.

Evaluation of Conventional and Molecular Techniques in Diagnosis of Clinical Samples of Mycobacterium Tuberculosis

In developing countries like Pakistan, rapid diagnosis and effective treatment in life threatening disease such as tuberculosis (TB) is very important. In the study 90 sputum samples from different hospitals were processed using auramine staining, Ziehl-Neelsen staining (ZN), culture on Löwenstein-Jensen (LJ) medium and Polymerase Cain Raction (PCR). Out of all 90 samples, 44 (48.88%) and 56 (62.2%) showed positive sputum smear microscopy, tested by Zeil Neilson and auramine staining respectively. Culturing into Lowenstein Jensen Medium revealed 64 (71.1%) and PCR yield highly significant 83 (92.2%) positive result. In conclusion, it was noted that PCR as a molecular technique is a very rapid, specific and sensitive method for diagnosis of TB as compared to other conventional methods.

Comparison of the culture and PCR methods for diagnosis of Mycobacterium tuberculosis in different clinical specimens

Clinical Biochemistry, 2011

Background: Tuberculosis remains a global epidemic, especially in developing countries, including Iran. Rapid diagnosis of active Mycobacterium tuberculosis infection plays a critical role in controlling the spread of tuberculosis. Conventional methods may take up to several weeks or longer to produce results. In addition to multiplicity of steps involved in conventional detection, including isolation, identification and drug susceptibility testing, the slow growth rate of M. tuberculosis is also responsible for this lengthy time. Objectives: The aim of this study was to compare the polymerase chain reaction (PCR) and culture methods for the detection of M. tuberculosis in different clinical specimens. Materials and Methods: This study was performed on different samples (urine, gastric aspirate, bronchoalveolar lavage, pleural fluid, cerebrospinal fluid, ascetic fluid and joint fluid specimens) of tuberculosis suspected patients. M. tuberculosis DNA was extracted directly from different samples using two different protocols. Next, PCR was performed using three sets of specific primers to detect members of Mycobacterium genus, M. tuberculosis complex and non-tuberculosis Mycobacteria. The results were then compared with that of the culture method, which is considered as the gold standard method. Results: The concordance rate between the three sets of primers was calculated and IS6110/buffer PCR method showed good agreement with the LJ culture method (κ = 0.627, P < 0.0001). The sensitivity of IS6110/buffer PCR was 58.33%, with specificity of 77.78%; the positive and negative predictive values were 100% and 78.26%, respectively. Buffer method for DNA extraction was proved to give a higher accuracy to PCR in comparison with the boiling method. Conclusions: PCR method is a valuable, cost-effective and alternative tool for quick diagnosis of active tuberculosis in different clinical specimens.

Sensitivity and Specificity of TaqMan Real Time PCR, PCR, Microscopy and Culture in Diagnosis of Tuberculous Meningitis in a High Incidence of Tuberculosis Province in Southeast of Iran

Biotechnology and Health Sciences, 2014

The most dangerous form of extra-pulmonary tuberculosis is tuberculous meningitis (TBM). Diagnosis of TBM has special problem due to its paucibacillary. Also, sensitivity and specificity of routine microscopy and culture in the diagnosis of this disease is controversial. So, faster and more accurate laboratory test is required. Polymerase chain reaction (PCR) and real time PCR may be good candidates for this purpose. Objectives: We did this study to compare sensitivity and specificity of TaqMan real time PCR, PCR, microscopy and culture in diagnosis of TBM. Patients and Methods: We had 49 patients with primary diagnosis of TBM during January 2007 and January 2008 in Bou-Ali University Hospital, Southeast of Iran. Combining and using the definite and probable TB as a gold standard, 29 of these patients had a final diagnosis of TBM. The extracted DNA of samples was applied for conventional PCR and TaqMan real time PCR. Results: Our study showed that the sensitivity and specificity of TaqMan real time PCR was 96% and 95% respectively. These values were 89% and 90, 38% and 100%, 6% and 100% for PCR, culture and microscopy, respectively. Conclusions: Our study showed that sensitivity of TaqMan real time PCR was higher that PCR, culture and microscopy but specificity of culture and microscopy was more than PCR and even TaqMan real time PCR.

Comparison of Culture and PCR Methods for Diagnosis of Mycobacterium tuberculosis in Different Clinical Specimens

Jundishapur Journal of Microbiology, 2014

Background: Tuberculosis remains a global epidemic, especially in developing countries, including Iran. Rapid diagnosis of active Mycobacterium tuberculosis infection plays a critical role in controlling the spread of tuberculosis. Conventional methods may take up to several weeks or longer to produce results. In addition to multiplicity of steps involved in conventional detection, including isolation, identification and drug susceptibility testing, the slow growth rate of M. tuberculosis is also responsible for this lengthy time. Objectives: The aim of this study was to compare the polymerase chain reaction (PCR) and culture methods for the detection of M. tuberculosis in different clinical specimens. Materials and Methods: This study was performed on different samples (urine, gastric aspirate, bronchoalveolar lavage, pleural fluid, cerebrospinal fluid, ascetic fluid and joint fluid specimens) of tuberculosis suspected patients. M. tuberculosis DNA was extracted directly from different samples using two different protocols. Next, PCR was performed using three sets of specific primers to detect members of Mycobacterium genus, M. tuberculosis complex and non-tuberculosis Mycobacteria. The results were then compared with that of the culture method, which is considered as the gold standard method. Results: The concordance rate between the three sets of primers was calculated and IS6110/buffer PCR method showed good agreement with the LJ culture method (κ = 0.627, P < 0.0001). The sensitivity of IS6110/buffer PCR was 58.33%, with specificity of 77.78%; the positive and negative predictive values were 100% and 78.26%, respectively. Buffer method for DNA extraction was proved to give a higher accuracy to PCR in comparison with the boiling method. Conclusions: PCR method is a valuable, cost-effective and alternative tool for quick diagnosis of active tuberculosis in different clinical specimens.

TB-PCR, UTILITY IN PRESENT SCENARIO - STUDY FROM TERTIARY CARE CENTER IN NORTHERN INDIA.

TBM is a syndrome of sub-acute lymphocytic meningitis in the majority of patients. When diagnosed promptly, TBM can be cured with supervised medication administration and supportive care. Even after many years of experience with the disease, the definitive diagnosis of TBM remains a problem. The nucleic acid amplification technique (NAA), notably the polymerase chain reaction (PCR) has revolutionized the investigative microbiology by facilitating direct detection and identification of infectious agent in the clinical samples in a very short time. AIM: Role of TB-PCR, in the diagnosis of tuberculous meningitis MATERIALS AND METHODS: This prospective study was done in Department of Medicine, and Department of Neurology over the one-year duration. The subjects were comprised of the patients above 10 years of age who got admitted with clinical picture commensurate with Meningitis were assessed on Thwarts Criteria and those found to be likely cases of T.B.M, as per the criteria were included in the study, and TB-PCR was done. RESULTS: Taking PCR positivity as the diagnostic test and evaluating its efficacy against Thwaites score <4, out of 99 cases of TBM, 42 were found to be true positive and 57 were false negative. CONCLUSION: Taking PCR positivity as the diagnostic test, out of 99 cases of TBM diagnosed by Thwaites score only 42.42% (n=42) were found to be true positive, hence it is not a good test for the diagnosis of Tuberculous meningitis.

Comparison of the conventional diagnostic modalities, bactec culture and polymerase chain reaction test for diagnosis of tuberculosis

Indian Journal of Medical Microbiology, 2005

Purpose: To evaluate the performance of 65 kDa antigen based PCR assay in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis. Methods: One hundred and fifty six samples were processed for detection of Mycobacterium tuberculosis by ZN smear examination, LJ medium culture, BACTEC radiometric culture and PCR tests. Results: A significant difference was seen in the sensitivities of different tests, the figures being 74.4% for PCR test, 33.79% for ZN smear examination, 48.9% for LJ culture and 55.8% for BACTEC culture (P<0.05). However, there was no significant difference (P>0.05) as far as specificity of different tests was concerned. PCR test sensitivity in pulmonary and extrapulmonary clinical samples were 72.7% and 75.9% respectively and found to be significantly higher (P<0.05) when compared with those of other tests. The mean detection time for M.tuberculosis was 24.03 days by LJ medium culture, 12.89 days by BACTEC culture and less than one day by PCR test. Conclusions: PCR is a rapid and sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.

Evaluation of Polymerase Chain Reaction and Cobas TaqMan Real Time PCR in the Diagnosis of Tuberculosis: Indian Prospective

Immunology and Infectious Diseases

Background & Objectives: Globally, tuberculosis (TB) still remains a major public health problem. India is a high TB burden country contributing to 26 per cent of global TB burden. Pulmonary tuberculosis (PTB) cases are more common (~ 90% of cases) while extra pulmonary tuberculosis (EPTB) constitutes around 10 to 20% of all tuberculosis cases in India. The diagnosis of the EPTB cases is difficult because of few bacilli and consequently is associated with low sensitivity of Zhiel-Neelson (ZN) smear and culture on LJ media. The present study evaluates the utility of PCR for the detection of M. tuberculosis in paucibacillary extra pulmonary and pulmonary tuberculosis samples. Methods: A total of 561 samples (553 EPTB & 8 PTB cases) were collected from the extra pulmonary and pulmonary tuberculosis patients which were processed for ZN smear, culture on LJ media and conventional PCR using two gene targets (IS6110 and MPB64). Results: The PCR positivity of IS6110 and MPB64 gene targets was found to be 91.3% (N=63/69) and 89.9% (N= 62/69) in majority of smear negative & culture positive (as a gold standard) extra pulmonary cases, respectively. However the PCR positivity was observed 100% in smear positive, culture positive Line probe assay tested MDR PTB cases (true positive controls; N=34). Further the PCR specificity was determined >95% (true negative healthy controls; N=26). The positivity of M. tuberculosis by IS6110 & MPB 64 gene targets was found to be range of 88% to 100% in various clinical paucibacillary extra pulmonary samples i.e. pleural fluid, ascitic fluid, lymph node, pus, CSF and others. Our data on 64 samples (non respiratory, n=63 & respiratory samples, n=1) revealed 40.6% positivity by Cobas TaqMan Real Time PCR (utilizing 16S rRNA probe; Roche, USA). Interpretation & Conclusion: Our data revealed that utility of both PCR and Real Time PCR in rapid diagnosis of M. tuberculosis in paucibacillary extra pulmonary tuberculosis samples in Indian scenario.