|Year : 2014 | Volume
| Issue : 2 | Page : 89-93
Evaluation of polymerase chain reaction using primer MPB 64 for diagnosis of clinically suspected cases of extrapulmonary tuberculosis
Dnyaneshwari P. Ghadage1, Vrishali A. Muley1, Sushma Pednekar2, Arvind V. Bhore1
1 Department of Microbiology, Smt. Kashibai Navale Medical College and General Hospital, Narhe Ambegaon (Bk.), Pune, Maharashtra, India
2 Department of Microbiology, B. J. Medical College and Sassoon General Hospitals, Station Road, Pune, Maharashtra, India
|Date of Web Publication||20-May-2014|
Vrishali A. Muley
Department of Microbiology, Smt. Kashibai Navale Medical College and General Hospital, Survey No. 49/1, 53/2, Off Mumbai-Pune Bypass, Narhe Ambegaon (Bk.), Pune - 411 041, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Pulmonary tuberculosis can be easily diagnosed by simple techniques such as microscopy. However, extrapulmonary tuberculosis (EPTB) often presents a diagnostic dilemma. Microscopy and culture have proved to be insensitive techniques for diagnosis of EPTB. There is an urgent need for rapid and sensitive diagnostic methods. Aim: The present study was conducted to evaluate the role of polymerase chain reaction (PCR) in the early diagnosis of clinically suspected cases of EPTB. Materials and Methods: A total of 80 clinical specimens comprising pleural fluid, cerebrospinal fluid, ascitic fluid, fine-needle aspiration biopsy, and pus and biopsy from clinically suspected EPTB cases were processed and followed up by conventional methods and PCR using MPB64 primer. Results: Tuberculous pleural effusion (71%) was found to be the most common clinical presentation of EPTB. Overall, PCR could detect EPTB in 61.2% cases. Microscopy and culture could detect 18.7% and 22.5% EPTB cases, respectively. PCR was positive in all tissue samples suggestive of tuberculosis on histopathological examination. Of the 62 EPTB patients who responded to antituberculosis treatment (ATT), 49 patients were PCR positive. Conclusion: PCR using MPB64 had a significant advantage over the conventional methods to detect the presence of M. tuberculosis in specimens of clinically suspected EPTB patients for early diagnosis of tuberculosis.
Keywords: Conventional techniques, extrapulmonary tuberculosis (EPTB), polymerase chain reaction (PCR)
|How to cite this article:|
Ghadage DP, Muley VA, Pednekar S, Bhore AV. Evaluation of polymerase chain reaction using primer MPB 64 for diagnosis of clinically suspected cases of extrapulmonary tuberculosis. J Sci Soc 2014;41:89-93
|How to cite this URL:|
Ghadage DP, Muley VA, Pednekar S, Bhore AV. Evaluation of polymerase chain reaction using primer MPB 64 for diagnosis of clinically suspected cases of extrapulmonary tuberculosis. J Sci Soc [serial online] 2014 [cited 2020 Sep 18];41:89-93. Available from: http://www.jscisociety.com/text.asp?2014/41/2/89/132837
| Introduction|| |
Tuberculosis has for centuries continued to be a public health problem of enormous importance, particularly in the developing world. In India, about 22 lakh cases of tuberculosis are added every year. The mortality rate is 5,00,000 per year, more than 1,000 per day, i.e., one every minute. Extrapulmonary tuberculosis (EPTB) contributes about 5-10% of the total cases of tuberculosis.  This apparent underestimation may be due to difficulty in diagnosis of EPTB, particularly with the unavailability of advanced technology in developing countries. In such a scenario, laboratory diagnosis of EPTB depends upon conventional methods such as microscopy and culture. Microscopy is an efficient technique for the diagnosis of pulmonary tuberculosis, but it lacks in sensitivity for diagnosis of EPTB. Isolation of Mycobacterium tuberculosis on culture is the gold standard for diagnosis but it requires a long period for growth. 
To overcome these limitations with conventional methods, quicker, specific, and sensitive diagnostic methods are being sought for the diagnosis of EPTB. Many gene amplification techniques for direct detection of M. tuberculosis are being attempted in research laboratories, with polymerase chain reaction (PCR) being one of them. Various species-specific primers are available for the detection of M. tuberculosis. One of these is the MPB64 antigen coding gene-based primer. The present study was conducted to find out the role of PCR using the MPB64 primer in the diagnosis of EPTB.
| Material and methods|| |
A total of 80 specimens from clinically suspected cases of EPTB were included in the study. Samples were collected before starting antituberculosis treatment (ATT) to the patient. Specimens were collected from patients of tuberculous pleuritis, tuberculous meningitis, tuberculous lymphadenopathy, cold abscess, and tuberculoma of breast. Specimens collected were cerebrospinal fluid (CSF), fine-needle aspiration biopsy (FNAB) sample, biopsy sample from tuberculoma breast, and pus sample from cold abscess. Each sample was divided into two parts. First part was used for microbiological investigations (microscopy and culture), and second part was stored at −20°C in a biofreezer until processed for PCR within 24 hours. Part of the tissue sample was sent to pathology laboratory for relevant cytopathological and histopathological investigations.
The samples were processed by standard microbiological procedure. , Smears were prepared and stained by Ziehl-Neelsen and Auramine 'O' fluorescent stain. Centrifuged deposit of CSF was directly inoculated on Lowenstein Jensen (L-J) medium. The pleural fluid, ascitic fluid, and FNAC/pus/homogenized tissue specimen were decontaminated by using modified Petroff's method and then cultured on L-J medium. Isolation on L-J medium was used as the gold standard for diagnosis of EPTB. The inoculated media were incubated at 37°C up to eight weeks. The slow-growing, non-pigmented, buff-colored, and dry colonies of acid-fast bacilli, accumulating niacin, reducing nitrate to nitrite were labeled as M. tuberculosis.
PCR testing was done in the molecular laboratory of the Microbiology Department of a medical teaching institute attached to a tertiary care hospital in Western India. DNA was extracted from 100 μl of the pellet from centrifuged deposit/100 μl of pus and FNAB samples by using silica gel method. , After DNA extraction, the samples were subjected to PCR. The MPB64 gene based primer (Source: Bangalore Genei, India) that gives 240-bp product was used for the amplification. An initial denaturation at 95°C for 3 minutes was performed to ensure complete separation of the two strands of template DNA. The thermal cycler was set for 30 cycles. Each cycle consisted of a step of denaturation at 95°C for 40 sec, annealing at 65°C for 45 sec, followed by an extension step at 72°C for 40 sec. A final extension cycle at 72°C for 4 min was performed to ensure complete extension of partially extended PCR product. Subsequently, agarose gel electrophoresis using 2% agarose was carried out for analysis of PCR product. The band at 240 bp was considered as a positive PCR reaction, with no band in negative control [Figure 1].
Well numbers 3, 4, and 5 showing positive PCR giving 240-bp product (using MPB64 gene-based primer)
Well no. 1- negative control, 2- positive control, 3- pleural fluid sample, 4- FNAB sample, 5- biopsy sample, and 6- molecular weight marker
The results obtained from PCR and conventional methods were compared and evaluated statistically by applying test of sensitivity and specificity, positive predictive value, and negative predictive value.
| Results|| |
The study group comprised 80 clinically suspected cases of EPTB. Majority of the patients were in the age group of 30-40 years. In all, 65% (52) of the cases were male, while 35% (28) of the patients were female. The clinical specimens included in this study were obtained from patients with different clinical conditions of EPTB. [Table 1] presents the clinical presentation and specimen-wise results of PCR.
|Table 1: Distribution of EPTB cases and specimen-wise PCR Result in EPTB cases|
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Tuberculous pleural effusion (71%) was the most common presentation of EPTB. PCR could detect tuberculosis in 61.2% clinically suspected EPTB cases. PCR positivity for the MPB64 gene was 100% in FNAB and biopsy specimens.
Two different staining techniques, Ziehl-Neelsen stain and Auramine 'O', were used for the detection of tubercle bacilli. [Table 2] shows the results of microscopy in this study group.
A total of 18.7% cases were positive by microscopy. Fluorescent technique could detect four additional cases. The results of PCR were compared with those of microscopy as shown in [Table 3].
PCR could detect 34 additional cases, which were negative by microscopy.
Isolation of M. tuberculosis was attempted on L-J medium. The colonies on L-J medium were identified as M. tuberculosis by conducting niacin and nitrate tests. [Table 4] shows the results of culture.
Culture could detect tuberculosis in 22.5% EPTB cases. Maximum culture positivity was seen in pus from cold abscess (33%), followed by pleural fluid (26.3%), FNAB (25%), and CSF (12.5%).
As shown in [Table 5], PCR detected 31 cases more than those by culture.
On comparison with culture as the gold standard, sensitivity and specificity of PCR was found to be 100% and 50%, respectively.
On the basis of clinical diagnosis, patients were started on ATT by the treating physician. The patients were reviewed after the completion of intensive phase of treatment or every three months for response to the treatment with respect to clinical improvement. A total of 62 patients responded to the ATT.
Using response to therapy as the gold standard, PCR technique was evaluated against conventional technique [Table 6]. Conventional techniques were considered positive when either smear or culture was positive.
|Table 6: Comparison of PCR and Conventional technique with response to ATT|
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PCR showed 79% sensitivity for the diagnosis of EPTB, whereas the conventional technique showed 33.8% sensitivity. The specificity of both techniques was found to be 100%. Thus, PCR performed much better than the conventional technique for diagnosis of EPTB.
| Discussion|| |
Tuberculosis remains a major world health problem, with global mortality of 1.6-2.2 million lives per year.  The problem is compounded further by the emergence of multi-drug resistant strain and co-infection with HIV. Extrapulmonary tuberculosis is now beginning to emerge from the shadows of its senior cousin pulmonary tuberculosis.  In the present study, male predominance was observed in the patients of EPTB (65% Vs 35%). Tuberculous pleural effusion is the most common exudative pleural effusion present in India compared to the West, where malignant effusions are more frequent.  Same observations were made in this study where tuberculous pleural effusion was the most common presentation of EPTB followed by tuberculous meningitis, abdominal Koch's, and cervical lymphadenopathy [Table 1].
Laboratory diagnosis of tuberculosis is based usually on traditional methods of microscopy by Ziehl-Neelsen or fluorescent staining, and on isolation of Mycobacteria as the gold standard. However, in case of extrapulmonary tuberculosis, the Ziehl-Neelsen stain, although rapid and inexpensive, lacks sensitivity in clinical specimens. The laboratory culture of M. tuberculosis requires a long period; therefore, clinical and therapeutic decisions have to be made before the laboratory diagnosis becomes available. Since the conventional microbiological techniques proved to be unsatisfactory for the diagnosis of EPTB, molecular methods have been used for the same. These techniques are more useful for the detection of microorganisms, which takes long period to grow on culture.
Different sets of primers have been used for M. tuberculosis PCR, of which the most commonly used primers are IS6110 and MPB64. However, recent reports say that isolation from some geographical areas such as the Indian subcontinent contains less copies of the insertion sequence compared with the 8-15 copies usually found in strains from most developed countries. Of the 124 strains of M. tuberculosis reported from South India, 42.7% showed single to no copies of IS6110.  As the number of copies of the target sequence is an important determinant of PCR sensitivity, it would be lower for strains having only a few copies of IS6110. A 240-bp region from the gene, coding MPB64 species-specific cell wall protein antigen has been reported to be highly specific for M. tuberculosis complex.  Therefore, in the present study, MPB64 gene-based primer giving 240-bp products was used for the amplification.
Overall, 61.2% cases of EPTB were diagnosed by PCR using MPB64 as a primer.[Table 1] Lilly et al. and Seth et al. have reported 35.2% and 85% PCR-positive EPTB cases for MPB64, respectively. , In the present study, specimen-wise analysis of PCR results showed the highest percentage of PCR positivity in FNAB and biopsy samples (100%), followed by pleural fluid (64.9%), CSF (37.5%), ascitic fluid, and pus (33%).[Table 1] Kesarwani et al. have reported 92% positivity of PCR on different tissues, while Nagesh et al. have reported 73.6% positivity of PCR on tissue specimens from EPTB cases. ,
Microscopy for demonstration of acid-fast bacilli remains the most cost-effective and rapid diagnostic method for tuberculosis. In the present study, Ziehl-Neelsen and Auramine 'O' staining techniques were used for the diagnosis. The overall microscopy-positive cases were 18.7%.[Table 2] Chan et al. reported 15% cases that were positive by direct microscopy for detection of EPTB. In the present study, on comparison of results of PCR with microscopy it was found that PCR could detect 34 additional cases, which were negative by microscopy.[Table 3] This shows that PCR is more sensitive than routine microscopy in detecting EPTB cases. Same observations were made by other workers. , There should be 10,000 bacilli present in the fluid specimen to be seen on direct microscopy. Low sensitivity of microscopy may be due to paucibacillary nature of infection in case of EPTB.
Although the culture is considered as the gold standard in diagnosis of EPTB, in the present study, it could detect EPTB in 22.5% cases.[Table 4] Mycobacteria could not be isolated from the remaining 77.5% of specimens, though they were incubated for up to 8 weeks. Therese et al. reported 4% culture positive rate in EPTB cases.  As evident from [Table 5], PCR picked up additional 31 cases that were culture negative, thus increasing the detectability by 38.7%. There should be 10-100 viable bacilli present in the sample for the culture to become positive.  In this study, low positivity of culture could be because of paucibacillary nature of infection or the use of only solid L-J media for cultivation.
Granulomatous inflammation has extensive differential diagnosis such as sarcoidosis, histoplasmosis, and chronic fungal infections. Therefore, PCR has the advantage of confirming the diagnosis of tuberculosis rather than relying on only histopathology. In the present study, 100% tissue specimens (three FNAB specimens from cervical lymphadenopathy and one biopsy sample from tuberculoma of breast), which were positive for tuberculosis on histopathological examination, were positive by PCR for MPB64. Kesarwani et al. have reported 92.1% sensitivity of histopathology in the diagnosis of tuberculosis from different tissue samples.  In the same study, PCR was found to be 97.8% sensitive.
In the present study, the patients were reviewed for response to the treatment with respect to clinical improvement. Out of 80 EPTB patients, 62 responded to ATT. Of these, PCR could detect 49 cases. However, PCR could not pick up 13 cases of EPTB who had responded to ATT. Chakravorty reported 17% and 33% false-negative PCR among pleural effusion and lymph node biopsies, respectively.  The false-negative PCR results could be because of
- Sampling error;
- Presence of PCR inhibitors such as blood, host proteins, and eukaryotic DNA, proteases, etc; and
- Inefficient extraction of DNA from the specimen.
Comparing the results of PCR with conventional technique using response to the treatment as the gold standard, the sensitivity of PCR was found to be 79%.[Table 6] PCR performed significantly better as far as sensitivity was considered. On comparing PCR using MPB64 primer with conventional technique, Martins et al. found 70% and 88% sensitivity and specificity, respectively, for the diagnosis of EPTB. 
PCR also offered the advantage of speed in obtaining results rather than waiting for culture. In the present study, this technique has proved its tremendous potential in routine diagnosis of EPTB.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]