|Year : 2016 | Volume
| Issue : 3 | Page : 181-186
Utility of polymerase chain reaction for detection of Mycobacterium tuberculosis in suspected cases of tuberculosis lymphadenopathy
Khushpreet Kaur1, KC Agarwal1, Ravindra Kumar2
1 Department of Pulmonary Medicine, Kamla Nehru Chest Hospital, Dr. S.N. Medical College, Jodhpur, Rajasthan, India
2 Central Research Laboratory, Sri Aurobindo Medical College and PG Institute, Indore, Madhya Pradesh, India
|Date of Web Publication||9-Jun-2016|
Central Research Laboratory, Sri Aurobindo Medical College and PG Institute, Indore, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Introduction: There is a need for a rapid and cost-effective technique for reliable diagnosis of tubercular lymphadenopathy, particularly in low-resource setting. In this study, we have used various diagnostic techniques including polymerase chain reaction (PCR) to diagnose clinically suspected cases of tubercular lymphadenopathy and compared the results to see which of the techniques are more sensitive, specific, and cost-effective. Materials and Methods: All patients having swelling in the neck, axillary, and inguinal regions were recruited for the study. Sputum for acid-fast Bacillus (AFB), fine-needle aspiration cytology, excision biopsy, DNA-PCR, AFB smear of the same material was done as per standard protocol. Results: 32% patients have granulomas with necrosis, whereas 30% have acute suppurative lesions and 24% and 14% patients were having only granulomas and only necrosis, respectively. A significant difference was observed between the PCR-negative and positive cases with respect to their cytomorphologic features. Positive AFB and tuberculosis-PCR (TB-PCR) results were significantly more common in the cases with chronic granulomatous inflammation in comparison to the cases showing chronic inflammation only. Sensitivity and specificity of TB-PCR were found to be 71.4% and 28.4%, respectively. Conclusion: PCR is a sensitive and rapid technique in the demonstration of Mycobacterium tuberculosis. It should be done in clinically suspected patients of tuberculous lymphadenopathy when their AFB stain is even negative.
Keywords: Fine-needle aspiration cytology, lymphnodepathy, polymerase chain reaction, tuberculosis
|How to cite this article:|
Kaur K, Agarwal K C, Kumar R. Utility of polymerase chain reaction for detection of Mycobacterium tuberculosis in suspected cases of tuberculosis lymphadenopathy. CHRISMED J Health Res 2016;3:181-6
|How to cite this URL:|
Kaur K, Agarwal K C, Kumar R. Utility of polymerase chain reaction for detection of Mycobacterium tuberculosis in suspected cases of tuberculosis lymphadenopathy. CHRISMED J Health Res [serial online] 2016 [cited 2019 Sep 22];3:181-6. Available from: http://www.cjhr.org/text.asp?2016/3/3/181/183735
| Introduction|| |
Tuberculosis (TB) - a disease caused by Mycobacterium tuberculosis bacterium, continues to be a public health challenge all over the world and in India for over 5000 years, and still continues to be a leading cause of morbidity and mortality. India is the highest TB burden country accounting for one-fifth of the global incidence. Global annual incidence estimate is 9.1 million cases out of which it is estimated that 1.9 million cases are from India. 
Extrapulmonary TB is one of the major problems in clinical practice. Children are more predisposed to develop extrapulmonary TB, which causes high morbidity.  In endemic areas, it is relatively common, in children to encounter tuberculous lymphadenitis, abdominal, and cutaneous TB which are difficult to differentiate - on clinical grounds - from nontuberculous conditions that need completely different lines of treatment. 
The most common form of extrapulmonary TB is tubercular lymphadenopathy  and its diagnosis remains a challenge since granulomatous lymphadenopathy has an extensive differential diagnosis.
Several conditions such as sarcoidosis, fungal infections, and other inflammatory conditions can present the same cytology and histopathology as tubercular lymphadenopathy. , The diagnosis of extrapulmonary TB is difficult, especially when clinical presentation is suggestive but bacteriological proof is lacking. The diagnosis confirmed by acid-fast Bacilli (AFB) using conventional microscopy is simple and rapid but lacks sensitivity, whereas culture is more sensitive and specific but takes several weeks to get the results. Fluorescent stain has been proven to be superior to the Ziehl-Neelsen's (ZN) stain, especially in paucibacillary cases.
Definitive and rapid diagnosis of extrapulmonary TB is challenging since conventional techniques have limitations. The chief difficulty with extrapulmonary specimens is that they yield very few Bacilli and consequently are associated with low sensitivity of AFB smear and culture. The diagnosis of extrapulmonary TB is challenging for a number of reasons: Lack of adequate sample amounts or volumes, apportioning of the samples for various diagnostic tests (histology/cytology/biochemical) analysis and microbiological and DNA-polymerase chain reaction [PCR]) resulting in nonuniform distribution of microorganisms, the paucibacillary nature of the specimens and lack of efficient sample processing techniques in extrapulmonary samples.
Traditionally, the diagnosis of tubercular lymphadenopathy is established by histopathology and smear microscopy or by mycobacterial cultures. Over the past decade, fine needle aspiration cytology (FNAC) has assumed an important role in the evaluation of peripheral adenopathy as a possible noninvasive alternative to excision biopsy. The cytological criteria for diagnosis of possible tubercular lymphadenopathy have been clearly defined as epitheloid cell granulomas with or without multinucleate giant cells and caseation necrosis. ,, About 20% have positive sputum cultures, but only 9-15% had positive AFB sputum smears. FNA biopsy leads to TB diagnosis in 79% of cases compared to surgical biopsy in 83%.  Nucleic acid amplification tests for TB lymphadenitis are rapid but yield variable and inconsistent results. 
Culture provides definitive identification, but it is time-consuming, requiring 6-8 weeks. Its efficacy in extrapulmonary TB is rather low. BACTEC system is capable of reducing the required time for isolation but has not been applied in most laboratories of our country. Positivity rates varying from 40% to 90% have been reported by DNA-PCR in the case of tubercular lymphadenitis.
In this study, we have used various diagnostic techniques including PCR to diagnose clinically suspected cases of tubercular lymphadenopathy.
| Materials and methods|| |
After taking into consideration the inclusion and exclusion criteria, 50 patients, either admitted or attending the outpatient clinic of our hospital who presented with lymphadenopathy and willing to participate in the study were enrolled after proper counseling. The protocol was explained to the patient or guardian (in the case of minor) before enrollment and informed consent was taken from each patient.
- Enlarged and palpable lymphadenopathy with diameter >1 cm
- No evidence of head and neck cancer
- No recent upper airway infection
- There was no spontaneous regression of cervical lymphadenopathy after 2 months of observation.
- Who treated with anti-TB drugs
- Have cancer
- Any systemic disease such as diabetes mellitus, primary immune deficiencies, or on long term steroids/immunosuppressive drugs.
Detailed clinical history was taken and physical examination carried out in all patients.
Patient selection was done on the basis of clinical suspicion of disease. Constitutional feature like chronic, low-grade fever, weight loss, anorexia, and enlarged palpable lymphnodes. All those patients having swelling in the neck, axillary and inguinal regions were considered and taken into consideration for further evaluation.
Battery of investigations like the chest X-ray posterior-anterior view, sputum for AFB, Mantoux test (0.1 mL volume containing 5 TU (tuberculin units), positive is taken when induration is (>10 mm after 48-72 h), FNAC, excision biopsy, DNA-PCR, AFB smear of the same material.
Routine blood test, liver function tests were performed as and when required.
Collection of sample
Lymph node was palpated and fixed. The area was cleaned with antiseptic and site was localized, 20 ml syringe was used to withdraw a sample. For PCR adequate amount was collected in vial containing 2 ml normal saline, and small amount was spread on a slide for AFB staining. The samples were sent to laboratory within 6 h of collection after proper labeling.
Fine-needle aspiration cytology
FNAC of lymph node was taken as suggestive of TB as evidenced by the presence of necrosis, hypocellularity, and epitheloid cells with or without giant cells and with or without AFB. FNA using a 23-25 gauge needle fitted to a 20 ml syringe was performed. After recording the gross appearance of the aspirate the syringes were spread on the slides and immediately transported to the laboratory where all samples were processed. Making smears of size 3 cm × 2 cm. One smear was fixed immediately with alcohol and stained with hematoxylin and eosin stains. The second slide was air dried, heat fixed stained by ZN method and observed for AFB.
Histopathology surgical (excisional) biopsy
Information regarding each patient was obtained. Biopsy was done under local anesthesia. The specimens were fixed in 10% formalin and then processed by tissue processing machine using the following schedule adopting 24-h scheduling. 5-μ thickness sections were obtained from each patient's block using rotary microtome. Specimen was sent to laboratory within 2 h of surgical removal. All specimens were formalin-fixed paraffin wax processed tissues. The sample was stained with staining procedure (hematoxylin and eosin).
Mantoux (tuberculin) test
The standard recommended tuberculin test, known as the Mantoux test, is administered by injecting a 0.1 mL volume containing 5 TU purified protein derivative (PPD) into the top layers of skin (intradermally, immediately under the surface of the skin) of the forearm. The injection is typically made using a Ό- to ½-inch, 27-gauge needle, and a tuberculin syringe. The tuberculin PPD was injected just beneath the surface of the skin. A discrete, pale elevation of the skin (a wheal) 6-10 mm in diameter should be produced when the injection is done correctly. A standard dose of 5 TU (0.1 mL) consists of an intradermal injection of 5 TU PPD is injected intradermally (between the layers of dermis) and read 48-72 h later. This intradermal injection is termed the Mantoux technique.
DNA extraction and polymerase chain reaction protocols
PCR procedure begins by heat-denaturation of a DNA sample into single strands. Next, two synthetic oligonucleotides complementary to the 3' ends of the target DNA segment of interest are added in great excess to the denatured DNA, and the temperature is lowered to 50-60°C. These specific oligonucleotides, which are at a very high concentration, will hybridize with their complementary sequences in the DNA sample whereas the long strands of the sample DNA remain apart because of their low concentration. The hybridized oligonucleotides then serve as primers for DNA chain synthesis in the presence of deoxynucleotides (dNTPs) and a temperature-resistant DNA polymerase such as that from Thermus aquaticus (a bacterium that lives in hot springs). This enzyme called Taq polymerase can remain active even after being heated to 95°C and can extend the primers at temperatures up to 72°C. When synthesis is complete, the whole mixture is then heated to 95°C to melt the newly formed DNA duplexes. After the temperature is lowered again, another cycle of synthesis takes place because the excess primer is still present. Repeated cycles of melting (heating) and synthesis (cooling) quickly amplify the sequence of interest. At each cycle, the number of copies of the sequence between the primer sites is doubled; therefore, the desired sequence increases exponentially - about a million-fold after 20 cycles - whereas all other sequences in the original DNA sample remain unamplified.
Those patients whose PCR was negative were subjected to excision biopsy of the nodes and proved histopathologically as TB.
Chi-square test is used in this research work for comparisons of different variables performed in our study.
| Results|| |
Total 60 patients were enrolled in the study. 50 patients finally completed the study as per protocol. Swelling was the only presentation in most of the cases 55%, evening rise of temperature in 22% patients and fever with a cough in 18% patients in addition to swelling.
The most common site of lymphadenopathy was found to be cervical region, i.e., 80% of the cases. Majority of the patients presented with nontender swelling in (76% cases). Firm, discrete swelling and periadenitis was present in 72% of the cases. The Monteux test was found positive in 72% of the patients. Chest X-ray was found normal in most of the patients (80%). Pulmonary involvement, in the form of mediastinal lymphadenopathy and parenchymal infiltrates, was observed in 14% and 6%, respectively. FNAC findings revealed that 32% patients showed granulomas with necrosis, whereas 30% had acute suppurative lesions 24% and 14% were found to have only granulomas and only necrosis, respectively [Table 1].
|Table 1: Correlation between tuberculosis polymerase chain reaction and various cytomorphological features of studied cases|
Click here to view
Significant difference was observed between the PCR-negative and positive cases with respect to their cytomorphologic features (P < 0.001). PCR positivity was found in 24 out of 28 (86%) cases showing granulomas with or without necrosis. Only 1 out of 7 cases (14%) was PCR positive which showed necrosis only.
Histopathological diagnosis was obtained from excisional biopsy of the lymph node. Chronic granulomatous inflammation was demonstrated in 70% of the patients whereas 30% patients showed only chronic inflammation without a definite evidence of granulomas.
Of 35 cases having chronic granulomatous inflammation, 7 cases (20%) were AFB positive and 25 cases (71%) were TB-PCR positive. Of the 15 cases of chronic inflammation, none were AFB positive and one (7%) was found positive by TB-PCR [Table 2].
|Table 2: Correlation between histopathologic diagnoses, acid-fast Bacillus stain results, and tuberculosis polymerase chain reaction results|
Click here to view
Positive AFB and TB-PCR results were significantly more common in the cases with chronic granulomatous inflammation in comparison to the cases showing chronic inflammation only. [Table 2] shows the sensitivity of TB-PCR 71.4% and specificity of TB-PCR 28.4%.
| Discussion|| |
In our study, most of the cases were in the age group of 0-30 years. Several reports have indicated a variable age group ranging from 18 months to 85 years. However, results in the present study are similar with the previous findings ,, where lymphadenopathy was most frequently found in children and young adults. Female preponderance was seen in our study. A positive family history of the TB was found in 20% of patients.
Most of the patients 80% presented lymphadenopathy of 0-12 month's duration. This observation is also found to be consistent with the previous studies. ,
Park et al. 2003  have also observed that out of the 81 patients studied 53 (65.4%) had chronic granulomatous inflammation, and 28 (34.6%) had only chronic inflammation on histopathology Ahmed et al. 2011  reported that 68% of the patients had strong evidence on histopathology (caseating granulomatous lesions), and 32% had noncaseating granulomas. In this study, granulomatous inflammation was observed in 70% of cases.
20% patients were found to be AFB smear positive in chronic granulomatous inflammation. Different studies have reported a wide range of AFB positivity ranging from as low as 0% to as high as 75%.  AFB positivity in smears and histological specimens depends on the bacillary load of the specimen and the type of the material. 
26 out of 50 patients (56%) were found to be TB DNA-PCR positive. Pahwa et al.,  have reported that over all PCR positivity was 40% in patients of tuberculous lymphadenopathy. Park et al.,  have also reported that of the 81 patients studied 46 patients were found to be tuberculous PCR positivity, i.e., (57%). The sensitivity of the PCR to detect M. tuberculosis in patients with clinical diagnosis of tuberculous lymphadenitis reported in the literature as 76.4-96%. However, positivity rates by DNA-PCR in the case of tubercular lymphadenitis varying from 40% to 90% have been reported by various authors. ,,
We have also correlated the results of tuberculous - DNA-PCR and the results of AFB smear staining. It was observed that out of the 26 cases that were tuberculous DNA-PCR positive 6 cases 23% were found to be AFB smear positive. On the other hand, out of the 7 AFB smear positive cases, 6 cases (86%) were tuberculous DNA-PCR positive.
Pahwa et al.  have also observed a higher positivity of DNA-PCR in cases with only granulomas (81.8%) followed by granuloma with necrosis only (57%) and the least in necrosis only (10%). In the present study, out of 35 cases having chronic granulomatous inflammation on histopathology, 20% patients were found to be an AFB smear positive and 71% patients were TB-PCR positive. On the other hand of the 15 cases of chronic inflammation without definite evidence of granulomatous inflammation, none was AFB positive, but 7% were TB-PCR positive.
Therefore, we observed that positive AFB and TB-PCR results were significantly more common in cases with chronic granulomatous inflammation than in the cases with chronic inflammation only. Furthermore, this difference was found to be statistically significant.
On the other hand, out of the 7 AFB smear positive cases, 6 cases (86%) were TB DNA-PCR positive. Similar observations have been reported by Park et al.  where 30% cases were AFB positive.
In our study, TB DNA-PCR was found to be positive, even in cases that were AFB negative of lymph node aspirate. The ability of DNA-PCR to detect AFB smear negative cases is remarkable as it can reduce the need for more invasive diagnostic approaches. These observations of our study are in agreement with Pahwa et al.  They reported that DNA-PCR was able to detect 80% of the smear negative but culture positive cases.
Pahwa et al.  have also observed a higher positivity of DNA-PCR in cases with only granulomas (81.8%) followed by granuloma with necrosis only (57%) and least in necrosis only (10%). Furthermore, there were 41% cases, that were smear positive and culture negative but DNA-PCR positive which is indicative of the fact that viable organisms are required for culture while genomic DNA can still be detected by PCR.
In the present study, we have investigated the relationship between histopathological diagnosis, AFB smear results and the results of TB-PCR for diagnosis of TB in lymphadenopathy.
We did not use mycobacterial culture of tissue specimens as a reference for the TB-PCR because corresponding specimens were not submitted for tubercular tissue culture. Although definite diagnosis of tubercular lymphadenopathy is achieved through bacterial culture, the sensitivity of mycobacterial isolation is very low and the isolation process takes a long time.
These results suggest that the classic histopathologic features of chronic granulomatous infiltration are compatible with the positive TB-PCR results in majority of cases. It is noteworthy that 7% cases with chronic inflammation but without definite granulomatous lesions were also positive by TB DNA-PCR. Similar observations have been made by Park et al.  They reported that a substantial proportion of cases 36% with chronic inflammation but without definite granulomatous lesion were also TB-PCR positive. They attributed these results to immunosuppression or the small size of biopsy specimens.
Up to now the gold standard for diagnosis of tuberculous lymphadenitis is the demonstration of mycobacterium from biopsy specimens by smear or culture. The sensitivity of mycobacterium isolation is very low and the isolation process takes a long time. The absence of specific cytologic findings of granulomatous lymphadenitis or negative AFB smear (80%) required additional open biopsy or repeated FNAC. Cytology lacks specificity due to the difficulty in establishing granuloma and other granulomatous pathologies in the absence of AFB. Thus, there was a need for a rapid and sensitive method for the diagnosis of tuberculous lymphadenopathy.
In terms of the speed of diagnosis the TB-PCR technique, the sensitivity of DNA-PCR was 71.42% and specificity is 28.4% is advantageous because sensitivity of AFB stain is low 20% and bacterial culture for TB is prolonged.
| Conclusion|| |
DNA-PCR is the sensitive minimally invasive and rapid technique in the demonstration of M. tuberculosis DNA in clinically suspected patients of tuberculous lymphadenopathy when their AFB stain is even negative.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]