Introduction

Tuberculosis (TB) continues to be a major health problem in developing countries. Lymphadenopathy is the most common presentation of extrapulmonary TB. ^[1,2]The clinical parameters for the diagnosis of TB in lymph nodes are neither specific nor do their absence exclude TB involvement. ^[3,4] Fine-needle aspiration cytology (FNAC) of lymph nodes in TB has varied cytomorphological features. However, the conventional Ziehl-Neelsen (ZN)  method for acid-fast bacilli (AFB) plays a key role in the diagnosis and also for the monitoring of treatment in TB. Its major disadvantage is low sensitivity ranging from 20% to 43%. ^[5,6]Mycobacterial culture is the reference method for the detection of tubercle bacilli but it is time consuming and requires specialized safety procedures in laboratories. Serological techniques have the disadvantage of lack of sensitivity and specificity. ^[5] Newer molecular techniques such as polymerase chain reaction (PCR), although rapid, are costly to be routinely used in developing countries where most TB cases occur. ^[7] Hence, a method for the identification of AFB which is more sensitive than the ZN method is required for early detection of TB. Although there are previous literatures related to the comparison of fluorescent microscopy with ZN stain for the detection of AFB in sputum in pulmonary tuberculosis ,but very few liteture available on efficacy of fluorescent method over conventional ZN method in extrapulmonary tuberculosis using samples like lymph node aspirates. Thus, the present study was undertaken with the following.

Aim : To study the efficacy and advantages of using fluorescent microscopy over conventional ZN method in lymph node aspirate to study AFB in suspected tuberculous lymphadenitis.

Objectives:

1.To study various cytomorphological features of lymph node aspirates in suspected tuberculous lymphadenitis and their association with detection of AFB on lymph node aspirate using conventional ZN stain under light microscopy and auramine-o stain under fluorescent microscopy. 

2.To correlate the fluorescent method with the conventional ZN method on lymph node aspirates for the detection of AFB in suspected tuberculous lymphadenitis.

Material and Methods

287 patients suspected clinically of having Tubercular lymphadenopathy referred for FNAC to the Department of Cytology from July 2014 to June 2015 were included in the study. Exclusion criteria were any lymphoid malignancy or metastasis, patients receiving anti-tubercular drugs and patient’s refusal to study. Relevant investigation details, such as hematology, chest radiogram, were reviewed in these patients. All the aspirates of  FNAC were processed for routine cytology, conventional ZN staining by direct microscopy and compared with the findings of the fluorescent method. A total of three or more smears were prepared from each of the FNAC aspirates: one alcohol-fixed wet smear stained by hematoxylin and eosin (H and E) for cytological examination, the second and third air-dried smear stained with ZN and AO stains, respectively for detection of mycobacteria. 

The following modified fluorescent staining procedure was implemented:

1.The heat-fixed smears were stained with the filtered Auramine-O mixture at 37°C for 15 minutes.

2.The slide was rinsed with de-iodinized water for 2 minutes.

3.Decolorization was performed with 0.5% hydrochloric acid in 70% ethanol for 2 minutes.

4.The slide was rinsed with deiodinized water for 2 minutes.

5.Counterstaining was performed with 0.5% aqueous potassium permanganate for 2 minutes.

6.The slide was rinsed with deiodinized water for 2 minutes, and air dried and examined under high power (×400) which was confirmed under oil immersion (×1000).

The AFB appear as yellow to orange, slender, rod-shaped bacilli under fluorescent microscopy. Smears stained by the conventional ZN method, directly, were examined for AFB under oil immersion (×1000) using light microscopy. 

The data were processed using test of association (chi-square test).

Results

A total of 287 fine-needle aspirated specimens from lymph nodes were aspirated during the study. Of these, 232 specimens were evaluated and the remaining 55 were eliminated because seven aspirates identified malignancy and three were inadequate. A total of 20 patients were HIV positive. 

The age ranged from 2 to 65 years, with the mean age of 26.22 years.

Female preponderance was noted accounting for 62% (144/232) of cases with male for 38% (88/232). 

Among the 232  lymph nodes studied, aspirates were from cervical (n = 192), axillary (n = 22), inguinal (n = 8), abdominal (n = 6)and multiple sites (n = 4) groups. 

Fig 1 : relative proportion of different sites

On cytomorphology, the tuberculous lymph node, was diagnosed using the following criteria: (i) purulent with caseation; (ii) only caseation; (iii) caseation with epithelioid cells; and (iv) noncaseating with epithelioid cells. ^[9]   

Fig. 2 : cytomorphological distribution of the cases

Table 1: The comparison of the results of the fluorescent method and the conventional ZN method with routine cytology is emphasized

Of the 116 aspirates, the smear positivity for AFB on the conventional ZN method was 35.34% (82/232) while the positivity increased to 63.79% (148/232) on the modified fluorescent method. The correlation between the conventional ZN method and the modified fluorescent method showed statistical significance (χ²= 18.7773, df = 1, P< 0.01).

Fig 3: comparative analysis between  ZN method and fluorescence method

Discussion

India has a long history of research and demonstration projects on TB. ^[10] The detection of AFB is often considered as the evidence of the infected state. Thus, the laboratory plays a critical role in the diagnosis of TB. ^[11] In developing countries, microscopy of the specimen is by far the fastest, cheapest, and most reliable method for the detection of AFB.

Since the early 1940s, the comparison of the fluorescent method with the conventional ZN method on sputum smears was implemented to improve the smear positivity for the detection of AFB. The use of a fluorochrome acid-fast stain, such as Auramine-O, is recommended because of its increased sensitivity and ease of interpretation compared with the ZN method. ^[12] The accepted practice is to stain sputum smears with AO at room temperature. ^[13-15] The present study was the first attempt to use the modified fluorescent method and compare it with the conventional ZN method on lymph node aspirates (in cytology) in this institute. The staining of these lymph node smears with AO at 37°C for 15 minutes increased the smear positivity over the conventional ZN method. Staining at 37°C is no more difficult than staining at room temperature and requires only that the AO stain be prewarmed prior to use. 

The AFB typically fluoresce as golden, slender, rod-shaped bacilli, but they may appear curved or bent. Also, some individual AFB may display heavily stained areas referred to as beads and/or alternating light and dark areas of stain producing a banded appearance. Although the ability to retain aryl methane dyes, such as Auramine O, after washing with alcohol or weak acids is a primary feature of the genus Mycobacterium, it is not entirely unique to the genus. Other bacteria, which contain mycolic acids, such as Nocardia, can also exhibit this feature. The exact method by which the stain is retained is unclear but it is thought that the stains become trapped within the cell or may form a complex with the mycolic acids. This is supported by the finding that shorter chain mycolic acids or Mycobacterial cells with disrupted cell walls stain weakly acidfast. A disadvantage is that there is a more intense binding of the mycolic acids to the fluorochrome dye causing bacilli, which are apparently rendered nonviable by chemotherapy to be acid-fast. ^[16,17] However, good observation is required to distinguish with certainty AFB from other small, naturally fluorescent particles present in some smears. When first using fluorescent microscopy, it is necessary to examine all small fluorescent objects seen both with the ×10 and ×40 objectives. With practice, it becomes possible to distinguish bacilli with a fair degree of certainty under the ×10 objective only, so that almost all negative smears can be examined with this objective only. However, it is always necessary to confirm the bacillary morphology with the higher power when the smears contain a low density of bacilli. Finally, if any doubt remains, it is possible to ring individual suspicious objects with the diamond objective marker, and restain the fluorescence stained smear by the ZN method to examine under an oil-immersion lens for cross-checking.

Previous studies on lymph node aspirate have shown the positivity rates for both the ZN and fluorescent methods as 44.11%  and 81.37%, respectively. ^[14] But, in our study on lymph node aspirates in cytology, the positivity rates were 35.34% for the ZN method and 63.79% for fluorescent method (P<0.001). 

We conclude that the  fluorescent method is more sensitive than the conventional ZN method. The advantage of using the fluorescent-stained smears is that, it can be examined  under  low  magnification allowing for much larger areas of the smear to be examined in a short period of time. The use of the fluorescent method greatly improves the diagnostic value especially in patients with a low density of bacilli that are likely to be missed on ZN-stained smears. Also, the use of AO staining alone could not be an alternative method  to conventional ZN staining. Hence, it could be beneficial when the  fluorescent method is used as an adjuvant along with clinical parameters and cytological features in lymph node aspirates.

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