|Year : 2012 | Volume
| Issue : 3 | Page : 342-345
Rapid culture diagnosis of tuberculous lymphadenitis from a tertiary care centre in an endemic nation: Potential and pitfalls
JS Verma1, I Dhavan2, D Nair1, N Manzoor3, D Kasana1
1 Department of Microbiology, VMMC and Safdarjung Hospital, 110 029, India
2 Department of Histopathology, VMMC and Safdarjung Hospital, 110 029, India
3 Department of Bio-Sciences, Jamia Millia Islamia University, 110 025, New Delhi, India
|Date of Submission||13-Sep-2011|
|Date of Acceptance||13-Apr-2012|
|Date of Web Publication||8-Aug-2012|
Department of Microbiology, VMMC and Safdarjung Hospital, 110 029
Source of Support: None, Conflict of Interest: None
In spite of low sensitivity and specificity, standard diagnostic algorithm recommends fine needle aspiration cytology (FNAC) and direct microscopic screening for acid-fast bacilli (AFB) for the routine diagnosis of tuberculous lymphadenopathy (LNTB). In this study, the diagnostic utility of liquid broth based automated culture (BacT/ALERT 3D) technique was assessed in comparison with conventional techniques in 89 clinically suspected tubercular lymphadenitis patients. 60% (n = 53) were positive by FNAC and 38.4% (n = 34) demonstrated AFB in smear examination. BacT/ALERT yielded isolation in 43.1% (n = 38) aspirates, confirming tubercular aetiology. We also found six paediatric culture-positive cases which showed negative outcome by both FNAC and smear. Thus, we conclude that culture by BacT/ALERT, may be used for faster yield of Mycobacteria in LNTB, especially in children. Additionally, this could also be used as a platform for further differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria (NTM) infection and for testing of anti-tubercular chemotherapeutic agents whenever drug resistance is suspected
Keywords: BacT/ALERT automation, extrapulmonary tuberculosis, FNAC, Lowenstein-Jensen media, tuberculous lymphadenopathy
|How to cite this article:|
Verma J S, Dhavan I, Nair D, Manzoor N, Kasana D. Rapid culture diagnosis of tuberculous lymphadenitis from a tertiary care centre in an endemic nation: Potential and pitfalls. Indian J Med Microbiol 2012;30:342-5
|How to cite this URL:|
Verma J S, Dhavan I, Nair D, Manzoor N, Kasana D. Rapid culture diagnosis of tuberculous lymphadenitis from a tertiary care centre in an endemic nation: Potential and pitfalls. Indian J Med Microbiol [serial online] 2012 [cited 2019 Sep 23];30:342-5. Available from: http://www.ijmm.org/text.asp?2012/30/3/342/99498
| ~ Introduction|| |
Tuberculosis (TB) is one of the major public health problems with a high mortality rate in India. It accounts for one-fifth of the global incidence and two-third of the cases in South East Asia.  Tuberculous lymphadenopathy (LNTB) is the most common manifestation of extrapulmonary tuberculosis (EPTB), especially in children. A recent survey from Revised National Tuberculosis Control Programme (RNTCP) showed that there were 219,945 (18% of total cases of TB) new cases of EPTB registered in the year 2008.  An Indian paediatric study showed the prevalence of peripheral lymphadenopathy as 27.2/1000 children and that of LNTB as 4.43/1000. 
In India, the incidence of mycobacterial lymphadenitis has increased in parallel with the increase in the incidence of HIV infection. The survey demonstrated that the prevalence of HIV among TB patients varied substantially across the geographical regions between 1% and 13.8%.  LNTB may present as a unilateral, single or multiple painless lumps, mostly located in the posterior cervical or supraclavicular region. Although Mycobacterium tuberculosis complex (MTC), i.e. Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium microti, is the main causative agent, a significant increase in the number of non-tuberculous mycobacterial (NTM) infection has been observed after the onset of HIV epidemic.  The environmental opportunistic mycobacteria (i.e. NTM) include over 100 species, many of which cause cervical lymphadenitis in children and pulmonary diseases and skin infections in adults. Causative species identification is always a clinician's concern as tuberculous lymphadenitis is best treated as a systemic disease with anti-tuberculosis medications whereas NTM infections can be addressed as local infections and are amenable to surgical therapy. Patients infected with NTM usually do not respond to conventional anti-tubercular treatment and are misdiagnosed as infection with multi-drug resistant strains of M. tuberculosis due to lack of correct species identification, particularly in the developing countries like India.
The diagnosis of LNTB is a challenge, especially when clinical evidence is suggestive but bacteriological confirmation is lacking.  Fine needle aspiration cytology (FNAC) being a simple outpatient diagnostic procedure is well accepted by patients and has practically no complications. The efficacy of FNAC as a diagnostic procedure has already been established and it has been found to be as efficient as biopsy, particularly in cases of LNTB, but it lacks specificity.  The diagnosis by direct microscopic screening of stained slides for acid-fast bacilli (AFB) is rapid and simple, but lacks sensitivity.  Also, now nucleic acid amplification techniques, notably the polymerase chain reaction (PCR), have been validated as rapid diagnostic tools,  but they are not a method of choice in regions with low-resource settings.
Bacterial isolation by culture is always considered as the gold standard in the diagnosis of M. tuberculosis. The user-friendly, simple and easy methodology makes it strongly recommended. Conventional solid Lowenstein-Jensen (LJ) medium usually requires 6-8 weeks before a positive result is obtained. However, with the introduction of mycobacterial enriched broth based BacT/ALERT system (bioMérieux Inc., Durham, NC, USA) method, the time required for recovery of mycobacteria from clinical samples has greatly reduced, along with significant sensitivity and specificity.  This assay utilises a colorimetric sensor and reflected light to monitor the presence and production of carbon dioxide dissolved in the culture medium. When micro-organisms present in the testing sample metabolise the substrate, carbon dioxide is produced. This changes the colour of the gas-permeable sensor installed in the bottom of each culture bottle from grey to light green or yellow. The colony forming units (CFUs) per milliliter at the time of detection is approximately 10 6 -10 7 .
| ~ Materials and Methods|| |
This study was conducted at a tertiary care centre, New Delhi, India, from January 2009 to June 2009, in which 89 (52 male and 37 female) clinically suspected patients with enlarged cervical and/or axillary lymph node(s) were enrolled after taking their informed consent. There were 23 (25.8%) paediatric cases, i.e. ≤17 years of age.
The FNAC (sample volume approx. 1.0-2.0 ml) was performed on the palpable lymph nodes of suspected cases of all the registered patients at the Department of Pathology according to the standard protocol, aseptically with sterile syringe and needle. After cytopathological examination, the remainder of the specimen, if scanty, was flushed into 1 ml of normal saline. These flushing materials were transferred to a cryovial aseptically, kept at −4°C, and cultured on LJ, automatic BacT/ALERT 3D Detection system.
The aspirated lymph nodes included cervical 67% (n = 60), supraclavicular 11% (n = 10), submandibular 6% (n = 5), auricular 11% (n = 10) and submental 5% (n = 4). A part of the aspirate was used for preparing two smears and stained by the Haematoxylin and Eosin (H and E) and by Ziehl-Neelsen (ZN) method as per the approved guidelines.  From the remaining portion, 0.2 ml aspirate was inoculated on LJ slant and 0.5 ml was put into BacT/ALERT enriched bottles for cultivation of bacilli. The growth on LJ slant was checked everyday in the first week to look for rapid growers, and thereafter on a weekly basis till the sixth week. The growth was finally screened by ZN stain for the presence of AFB. Similarly, once the BacT/ALERT system flashed a positive signal, smear was made from the broth and screened to confirm TB bacilli. The final diagnosis was made if there was epithelial cell granuloma with necrosis, and/or smear positivity for AFB and/or positive cultivation were seen. 
| ~ Results|| |
Of the 89 cases screened, 59 cases were collectively diagnosed positive for tubercular infection as per the protocol.  In this, 60% (n = 53) were positive by cytological examination and 38.4% (n = 34) demonstrated AFB in smear examination. BacT/ALERT automation yielded positive isolation in 43.1% (n = 38) aspirates, whereas LJ method showed 32.6% (n = 29) cases of tubercular aetiology [Table 1]. Considering culture as a gold standard, we found sensitivity, specificity, positive predictive value (PPV), i.e. ability to detect true positive, and negative predictive value (NPV), i.e. ability to detect true negative, of FNAC to be 60%, 49%, 56% and 65%, respectively, in adult cases [Table 2] and 40%, 75%, 60% and 66.6%, respectively, in paediatric cases [Table 3]. ZN-stained smear screening showed sensitivity, specificity, PPV and NPV of 50%, 70%, 51% and 66%, respectively, in adult cases and 33.3%, 78%, 50% and 63%, respectively, in paediatric patients. Chi-square statistical analysis showed significant value (P value < 0.05) in the paediatric cases evaluated. Six paediatric cases which showed negative diagnosis by both FNAC and smear examination were positive by culture.
|Table 1: Performance of all diagnostic parameters for different site specific aspirates|
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|Table 3: Performance of other tests in comparison to culture in paediatric cases|
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| ~ Discussion|| |
In FNAC, diagnosis is based on the presence of granulomas, central necrosis (suggestive of LNTB), and if possible, demonstration of AFB by staining of tissue sections (positive LNTB). Cytology has a sensitivity of approximately 32-59%. , Several reasons could be responsible for variation in FNAC outcome, i.e. scanty bacilli in glands, poor representation or inadequate sampling, and lesser cases of TB abscesses. ,, The major limitation identified in the FNAC was the small volume of material that could be aspirated.
The concentration of organisms in the clinical tubercular specimen has a direct relationship with the sensitivity of the ZN stain and a concentration of ≥10 4 organisms/ml is needed to show a positive smear. The overall AFB positivity in fine needle aspiration smears can vary from 37.4 to 59.4%. , In the present study, it was 38.4% of the total cases and 50% of all culture-positive aspirates. The low sensitivity was probably due to the low concentration of mycobacteria in the aspirate. Few investigators showed that Universal Sample Processing (USP) technology method can improve the detection rate. 
There were nine discordant isolates between BacT/ALERT and LJ, which were again sub-cultured from liquid broth on LJ media and showed positive result this time. The reason might be attributed to low bacterial load and non-uniform distribution in the aspirated sample. The detection time of BacT/ALERT was between 8 and 24 days, i.e. median value was ≤3 weeks, whereas LJ method took 6 weeks. Culture reports from different studies detected fine needle aspirates between 39 and 80% positive in the clinically suspected LNTB cases. ,, Our observation also falls within the reported range, i.e. 43.1%. Also, there were significant (16.8%) false-negative results observed by culture as compared to cytology results. This might be attributed to fractionation of the sample for various diagnostic tests like cytology and the microbiological parameters resulting in non-uniform distribution of micro-organisms. There may also be a possibility for the presence of nonviable bacilli due to pre-exposure to broad-spectrum antibiotics such as amoxicillin-clavulanic acid and fluoroquinolones, which have been reported to inhibit M. tuberculosis leading to negative culture.  The most important outcome of the study was to rule out positive diagnosis in six paediatric cases in which both FNAC and smear findings were negative. This shows the potential and usefulness of culture in suspected paediatric cases of LNTB where repeat sampling is a big problem. A positive culture growth can be used further for species identification as well as differentiation of M. tuberculosis from NTM and the selection of anti-tubercular drugs whenever drug resistance is suspected.  So, missed cytological diagnosis and increasing prevalence of NTM justifies the incorporation of mycobacteria cultivation along with cytology in all suspected cases of LNTB, especially in a region of high prevalence like India.  The only concern about using BacT/ALERT automation in the routine lab settings of a developing nation is its cost effectiveness. A single test by BacT/ALERT costs approximately Rs. 200 (only cost of the bottle is estimated), This is significantly higher than that of conventional culture and fine needle aspiration cytology (which individually costs < Rs. 25/-).
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[Table 1], [Table 2], [Table 3]
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