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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 4  |  Page : 714-717
Efficacy of an in-house polymerase chain reaction assay for rapid diagnosis of Mycobacterium tuberculosis in patients with tubercular lymphadenitis: Comparison with fine needle aspiration cytology and conventional techniques


1 Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012, India
2 Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012, India
3 Department of Cytology, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012, India

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Date of Web Publication27-Oct-2010
 

   Abstract 

Introduction: Tubercular lymphadenitis (TB-L) is the most common manifestation of extrapulmonary tuberculosis. Excisional biopsy with histopathological examination, Ziehl-Neelsen staining (ZNS) and culture and fine needle aspiration (FNA) cytology, although useful in the diagnosis of TB-L, cannot diagnose a substantial proportion of cases. We investigated the role of an in-house polymerase chain reaction (PCR) assay targeting the IS6110 gene from the FNA material in the diagnosis of the disease. Materials and Methods: The clinical profile of 150 patients with lymphadenopathy was noted and the fine needle aspirate was collected. After cytological processing, ZNS and culture on Lowenstein-Jensen media, mycobacterial DNA was isolated from the residual aspirate material and IS6110 gene PCR was performed. Results of cytology, ZNS, culture and IS6110 gene PCR were compared. Results: There were 49 confirmed patients of TB-L based on laboratory parameters (either culture isolation of Mycobacterium tuberculosis or any two of cytology, ZNS, PCR positive) and clinical response to therapy. Sensitivity and specificity of FNA was 89.8% and 96%, of ZNS was 40.8% and 99%, of culture was 40.8% and 100% and of IS6110 gene PCR test was 100% and 92.1%. Conclusion: IS6110 PCR can be considered a valuable adjunct to cytology, ZNS and culture techniques in the diagnosis of TB-L.

Keywords: Fine needle aspiration cytology, IS6110 PCR, tubercular lymphadenitis

How to cite this article:
Sharma M, Sethi S, Mishra AK, Chatterjee SS, Wanchu A, Nijhawan R. Efficacy of an in-house polymerase chain reaction assay for rapid diagnosis of Mycobacterium tuberculosis in patients with tubercular lymphadenitis: Comparison with fine needle aspiration cytology and conventional techniques. Indian J Pathol Microbiol 2010;53:714-7

How to cite this URL:
Sharma M, Sethi S, Mishra AK, Chatterjee SS, Wanchu A, Nijhawan R. Efficacy of an in-house polymerase chain reaction assay for rapid diagnosis of Mycobacterium tuberculosis in patients with tubercular lymphadenitis: Comparison with fine needle aspiration cytology and conventional techniques. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Dec 11];53:714-7. Available from: http://www.ijpmonline.org/text.asp?2010/53/4/714/72049



   Introduction Top


Tubercular lymphadenitis (TB-L), commonly referred to as scrofula or king's evil, is the most common manifestation of extrapulmonary tuberculosis. [1],[2] Asians, typically Indians, seem pre-disposed to lymph node involvement. [1],[2] A seemingly gradual, painless enlargement of the lymph nodes progressing over months is the typical presentation. [1],[2],[3] A similar condition can result from a host of other diseases, including malignant ones. [1],[2],[3] Rapid and accurate diagnosis of TB-L results in cure of the patient, which may otherwise lead to considerable morbidity. [1]

Traditional diagnosis hinges on clinical judgment, including history of contact with tuberculosis patients, tubercular skin testing (TST), abnormal chest radiographs excisional biopsy with histopathological examination, Ziehl-Neelsen staining (ZNS) and culture of the excised lymph node and finally, on response to antitubercular therapy. [1],[2],[3] Fine needle aspiration (FNA) of the lymph nodes with cytological, microbiological smear (ZNS, Kinyoun and auramine staining) and culture examinations have simplified diagnosis to a great extent. [1],[2],[3],[4] However, none of these methods alone can diagnose all cases of TB-L and there exists a need for a rapid and cost-effective technique for reliable diagnosis of TB-L, particularly in resource-poor settings. [4],[5],[6],[7] Earlier workers have devoted attention to the role of polymerase chain reaction (PCR) in the diagnosis of TB-L from FNA material. [4],[5],[6],7] However, no clear guidelines have as yet been formulated. In this study, an in-house PCR assay along with other diagnostic techniques was used to diagnose clinically suspected TB-L.


   Materials and Methods Top


A total of 150 patients (adults and children) who presented to the Outpatient Department or were admitted in our tertiary care center with clinical suspicion of lymphadenopathy were enrolled for the study. An informed consent was taken from each patient. A detailed history and physical examination was carried out and data regarding age, sex, lymph nodes involved, matted or discrete, history of contact with a diagnosed case of tuberculosis, relationship with the case, previous treatment for tuberculosis and presenting complaints were recorded. They were also screened for human immunodeficiency virus (HIV) status as per the latest guidelines of NACO, India. [8]

FNA, using a 23-25-gauge needle fitted to a 20-mL syringe was performed on the cases by an experienced pathologist. After recording the gross appearance of the aspirate, the syringes were immediately transported to the laboratory. All samples were processed in Biosafety level II cabinets; drops of the aspirate material were expressed and spread on glass slides, making smears of size 3-4 cm X 2 cm. One smear was fixed immediately with alcohol and stained with hematoxylin and eosin stain. Two independent observers recorded their cytological findings. Epitheloid cell granulomas with or without multinucleated giant cells and caseation necrosis was the cytological criteria for diagnosis of TB-L. [6] A second slide was air-dried, heat-fixed, stained by Ziehl-Neelsen method and observed for acid fast bacilli (AFB).

From the FNA material left, two further aliquots were made, one for culture and the other for PCR. Culture was performed on Lowenstein-Jensen media and incubated at 37ºC for 8 weeks. Cultures were checked daily during the first week, then weekly thereafter. All positive cultures were confirmed for acid fastness by ZNS. Further identification was carried out using culture characteristics, growth on MacConkey Agar, rate of growth, biochemical reactions, including niacin test, nitrate reduction test, semi-quantitative catalase test and heat-s table catalase test.


   Polymerase Chain Reaction Top


Mycobacterial genomic DNA was extracted from the FNA samples as described previously. [9] A 123 bp fragment of IS6110 gene was amplified using primers IS1 and IS2. PCR conditions were standardized following previous reference. [10]

IS1 5'- CCTGCGAGCGTAGGTCGG-3'

IS2 5-'CTCGTCCAGCGCCGCTTCGG-3'

Amplification reaction was performed in a thermocycler (Techne® Genius Thermocycler, Cambridge, UK). DNA 2 μl, 2.5 μl 10X buffer, 0.5 μl of Taq DNA polymerase, 1 μl dNTP mixture and 1 μl of each primer (Sigma) was added to 18 μl of sterile double-distilled water (DDW) to obtain the reaction mixture. The reaction conditions were as follows: initial denaturation at 95ºC for 5 min, followed by 32 cycles of denaturation at 95ºC for 30 s, annealing at 58ºC for 1 min and extension at 72ºC for 1 min, and final extension at 72ºC for 10 min. [10] Positive control DNA of standard strain (H37RV) and negative controls (sterile DDW) were used in each run. PCR products were analyzed by agarose gel (1%) electrophoresis and ethidium bromide staining. Standardization of the PCR test was performed before the start of the study and involved repeated performance of the test from genomic DNA of the standard strain (H37RV), other known Mycobacterium tuberculosis isolates (isolated in our laboratory), known non-tuberculous mycobacterial isolates (isolated in our laboratory) and sterile distilled water. It was only when technical expertise was achieved (similar and valid results in three separate runs) that the test was carried out on study samples.


   Statistical Analyses and Gold Standard of Diagnosis Top


Although culture isolation of M. tuberculosis is the gold standard for diagnosis, none of the methods of laboratory diagnosis alone has 100% sensitivity to diagnose TB-L. Thus, our case definition for TB-L was: (1) positive culture for M. tuberculosis or (2) if culture was negative, then any two of the three other laboratory tests should be positive (acid-fast staining, FNA cytology showing epitheloid cell granulomas with or without multinucleated giant cells and caseation necrosis and PCR test). In addition, response to standard antitubercular therapy instituted on these patients was checked over a period of 12 months, and it was found that all 49 such patients responded to therapy with regression of lesions and absence of any systemic symptoms (fever, cough, weight loss). Statistical analysis was carried out using the software SPSS, version 15.0.


   Results Top


Forty-nine patients fulfilled our case definition of TB-L. The clinic-epidemiological and cytological details of the 49 patients with confirmed TB-L and the total 150 suspected cases are depicted in [Table 1]. Age of TB-L patients ranged from 3 to 76 years; males predominated (1.17:1), family history of tuberculosis was given in seven cases (14.3%) while past history of tuberculosis was present in eight cases (16.3%). Only seven of the 49 cases were children below the age of 14 years (14.3%), 32 in the age group of 15-40 years (65.3%), nine between 41 and 60 years (18.4%) and one patient was over 60 years of age (2%). Cervical lymphadenopathy was the most common (83.7%), followed by axillary (10.2%), abdominal (4.1%) and mediastinal (2%). HIV infection was diagnosed in six cases of TB-L (five cervical and one abdominal, all males, age - 22-68 years); of them, three were ZNS positive (50%), five cytology positive (83.3%) and all six PCR positive (100%).
Table 1 :Clinical, epidemiologic and cytology findings in patients with lymphadenitis (n = 150) and tubercular lymphadenitis (n = 63)


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In all, 48 cases showed epitheloid cell granulomas with or without multinucleated giant cells and caseation necrosis, 21 were positive on ZN smear examination while 20 were culture-positive for Mycobacterium spp. They were all later identified as M. tuberculosis complex. PCR [Figure 1] was positive in 57 samples. The results of culture, ZNS, cytological and PCR studies are summarized in [Table 2].
Table 2 :Results of tuberculosis detection by culture, ziehl-neelsen staining, cytology and polymerase chain reaction in cases of lymphadenitis*


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Figure 1 :Amplification of 123 bp DNA fragment of IS6110 element. Lane M: molecular weight marker; Lanes 1 and 4-8: clinical samples; Lane 2: positive control; Lane 3: negative control

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   Discussion Top


TB-L is a potentially fatal extrapulmonary manifestation of tuberculosis that can be effectively treated once definite diagnosis is established. [1],[2],[3] Differential diagnosis includes neoplasms (non-Hodgkin's lymphoma, Hodgkin's disease, sarcoma and metastatic carcinoma), infections (viruses - infectious mononucleosis, Chlamydia, bacteria including Streptococcus, fungi including histoplasmosis and cryptococcosis and Toxoplasma), drug reactions (phenytoin), sarcoidosis, non-specific reactive hyperplasia and non-lymphoid neck swelling. [1] The proportion of TB-L among extrapulmonary tuberculosis is slowly increasing. [1] PCR testing directly from clinical samples has revolutionized diagnosis in many areas of microbiology, especially in those of viral diseases. However, clear-cut guidelines regarding the usefulness of PCR in tuberculosis diagnosis are only in their stage of infancy. This is because of the potential problem of inhibitors in PCR testing from non-sterile samples, like sputum and broncho-alveolar lavage. However, PCR from aspirate samples can be helpful to establish a diagnosis of extrapulmonary tuberculosis, especially that of TB-L. [1],[2],[3],[7] In addition to smears, it can be used as a rapid diagnostic technique giving results within 5 h. In our study, 29 cases were ZNS and culture negative but PCR positive. These patients showed marked improvement on anti-tubercular therapy. Other studies too have documented the high sensitivity (90%) of PCR in the diagnosis of TB-L; Sekar et al. [7] detected 20 additional cases that were smear and culture negative. In addition, the PCR test did not miss any of the smear-, culture- or cytology-positive cases. However, PCR technology being costly, and requiring skilled manpower, cannot be used just as an adjunct to diagnosis. However, unlike respiratory samples, where the quantity of sample is large and can be repeated without discomfort, aspirates from lymph nodes are small in quantity and require invasive procedure. Thus, testing by PCR on the residual sample after all conventional diagnostic procedures have been performed may be a reasonable alternative.

Our in-house PCR targeted the multi-copy IS 6110 insertion sequence. IS 6110 belongs to the IS3 family of insertion sequences and is specific for the M. tuberculosis complex. Further, most strains carry 10-15 copies of the gene over the whole genome. [9] It may be absent in a few strains or may be present in low copy numbers when detection from clinical samples becomes difficult. [7] However, this may not be a major hindrance as earlier studies from India have shown IS6110 detection to be more sensitive for the diagnosis of tuberculosis. [5],[6],[7],[10] In fact, Negi et al. [11] reported IS6110 detection (77%) to be more sensitive than PCR targeting 65 kDa (75%), 38 kDa (72%) and 85B protein (73%).


   Conclusion Top


Addition of IS 6110 PCR can be considered a valuable adjunct to cytology, ZNS and culture techniques in the diagnosis of TB-L.

 
   References Top

1.Powell DA. Tuberculous Lymphadenitis. Schlossberg Tuberculosis and Nontuberculous Mycobacterial Infections. 4th ed. Philadelphia, USA: W.B Saunders Company; 1999. p. 186-94.  Back to cited text no. 1
    
2.Iseman MD. Extrapulmonary tuberculosis in adults. A clinician's guide to tuberculosis. Philadelphia, USA: Lippincott Williams and Wilkins; 2000. p. 145-98.  Back to cited text no. 2
    
3.Bayazύt YA, Bayazύt N, Namidurub M. Mycobacterial Cervical Lymphadenitis. ORL J Otorhinolaryngol Relat Spec 2004;66:275-80.  Back to cited text no. 3
    
4.Khan RA, Wahab S, Chana RS, Naseem S, Siddique S. Children with significant cervical lymphadenopathy: Clinicopathological analysis and role of fine-needle aspiration in Indian setup. J Pediatr (Rio J) 2008;84:449-54.  Back to cited text no. 4
    
5.Singh KK, Muralidhar M, Kumar A, Chattopadhyaya TK, Kapila K, Singh MK, et al. Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy. J Clin Pathol 2000;53:355-61.  Back to cited text no. 5
    
6.Pahwa R, Hedau S, Jain S, Jain N, Arora VM, Kumar N, et al. Assessment of possible tuberculous lymphadenopathy by PCR compared to nonmolecular methods. J Med Microbiol 2005;54:873-8.  Back to cited text no. 6
    
7.Sekar B, Selvaraj L, Alexis A, Ravi S, Arunagiri K, Rathinavel L. The utility of IS6110 sequence based polymerase chain reaction in comparison to conventional methods in the diagnosis of extra-pulmonary tuberculosis. Indian J Med Microbiol 2008;26:352-5.  Back to cited text no. 7
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8.Guidelines on HIV testing. March 2007. National AIDS control authority. Ministry of Health and Family Welfare. Government of India.  Back to cited text no. 8
    
9.Van Soolingen D, Hermans PW, Hass De PE, Soll DR, Embden JD. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex: Evaluation of insertion sequence dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 1991;29:2578-86.  Back to cited text no. 9
    
10.Rodrigues C, Nukula R, Menon S, Hakimiyan A, Mehta AP. DNA amplification of IS6110 in rapid detection of Mycobacterium tuberculosis. Indian J Med Microbiol 1997;4:167-71.  Back to cited text no. 10
    
11.Negi SS, Anand R, Pasha ST, Gupta S, Blasir SF, Khare S, et al. Diagnostic potential of IS6110, 38 kDa, 65 kDa and 85B sequence based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples. Indian J Med Microbiol 2007;25:43-9.  Back to cited text no. 11
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Correspondence Address:
Sunil Sethi
Associate Professor, Department of Medical Microbiology, PGIMER, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.72049

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