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Microscopy detection of acid fast bacilli (AFB) by Ziehl–Neelsen (ZN) method has many advantages when it comes to speed and feasibility though it has a low sensitivity. If the sensitivity could be improved, it has the potential to become an even more valuable tool for detection of AFB.
To evaluate the efficacy of bleach concentration method in the cytodiagnosis of tuberculous lymphadenitis in comparison with routine Ziehl–Neelsen method and to compare the positivity in various cytomorphological categories.
A total of 112 cases of tuberculous lymphadenitis diagnosed by fine needle aspiration cytology (FNAC) were categorized into six cytomorphological patterns. The acid-fast bacilli positivity by routine staining was correlated with modified bleach methods of ZN staining. Sensitivity of routine ZN and modified bleach concentration was compared.
The classic cytomorphological pattern of tuberculosis of epithelioid granulomas, langhans giant cells and caseous necrosis was seen in 37.5% of cases. Routine ZN staining detected AFB in 12.5% of cases and the modified bleach method in 60.7%. Modified bleach method showed AFB positivity in additional 54 cases where routine AFB staining was negative. The modified bleach method showed AFB positivity in all cases where routine ZN staining was positive.
The modified bleach method was more sensitive and safer than routine ZN staining. As the background was clear, the bacilli were easily visible and the screening time was shorter.
Ziehl–Neelsen (ZN) method for acid-fast bacilli (AFB) plays a key role in the diagnosis and also for the monitoring of treatment in tuberculosis.[1,2] Its major disadvantage is low sensitivity,[3,4] ranging from 9 to 46%. Mycobacterial culture is the reference method for 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. Newer molecular techniques, such as polymerase chain reaction (PCR), are costly to be routinely used in developing countries where tuberculosis is prevalent.
Microscopy has many advantages when it comes to speed and feasibility, and if its sensitivity could be improved, it has the potential to become an even more valuable tool for tuberculosis control programmes around the world. The need for new, improved, low-cost techniques cannot be overemphasised. The bleach concentration method is one of the safest concentration methods for improving the sensitivity of direct microscopy for the detection of AFB. A few previous studies have shown that liquefaction of sputum by sodium hypochlorite (NaOCl, bleach) and concentration of bacilli through centrifugation will significantly increase the sensitivity of direct microscopy.[7,8]
In the present study, fine needle aspiration cytology (FNAC) was used to diagnose cases of tuberculous lymphadenitis. We used the bleach concentration method on fine needle aspirates and compared the sensitivity of this technique in detecting AFB with that of routine ZN staining on FNA smears. This study has been taken up to validate the studies done earlier.[9,10]
The study included 112 cases of tuberculous lymphadenitis diagnosed on the basis of cytomorphological features of smears in lymph node aspirates. FNA was performed with a 23-gauge needle and a 10-mL disposable syringe fitted with a syringe holder. Following lymph node aspiration, approximately 5–6 smears were made on clean glass slides. One or two smears were fixed in 95% ethanol and stained with Papanicolaou stain. Air-dried smears were stained using May-Grünwald–Giemsa stain (MGG) and the routine ZN method. The residual aspirated material in the needle was flushed out and subjected to liquefaction with 5% sodium hypochlorite (NaOCl, Hypo, bleach) solution in a test tube at room temperature for 30 min. The aspirated material along with the bleach solution was then centrifuged for 15 min at 3000 rpm. The supernatant was discarded and smears were made from the sediment on clean glass slides, followed by ZN staining. The slides were mounted and screened.
The spectrum of cytomorphological features observed on Papanicolaou- and MGG-stained smears was broadly categorized into six patterns using the following criteria: Epithelioid granulomas with langhans giant cells and caseous necrosis (pattern 1); epithelioid cells in a reactive background (pattern 2); caseous necrotic material with epithelioid cells (pattern 3); caseous necrotic material with a few lymphocytes and histiocytes; no epithelioid cells (pattern 4); only caseous necrosis (pattern 5); and smears with predominantly neutrophils and degenerating epithelioid cells and semifluid necrotic material (pattern 6).
The cervical group of lymph nodes was most predominantly affected in 90.2% of cases. Seven cases showed axillary lymph node involvement (6.25%) and two cases had inguinal lymph node involvement (1.75%). Two cases presented with generalized lymphadenopathy (1.75%). Most of the cases had fever, loss of weight, loss of appetite, and three patients had hepatosplenomegaly. Sixteen cases were positive for HIV.
Cytomorphological features of all the cases were analyzed based on the pattern. Pattern 1 was seen in 42 (38%) cases, pattern 2 in 27 (24%), pattern 3 in 8 (7%), pattern 4 in 17 (15%), pattern 5 in 2 (2%) and pattern 6 in16 (14%) cases.
Routine ZN staining detected 14 (12.5%) cases of AFB positivity and the bleach method detected 68 (60.7%) cases of AFB positivity. Hence, the modified bleach method detected 54 more cases than were detected by routine ZN staining.
None of the cases diagnosed positive on routine ZN stain was missed by bleach method. AFB positivity by bleach method was more in comparison with the routine ZN stain in all patterns [Figure 1].
In 16 patients with positive HIV serology, the predominant morphology was type 6 (31.2%), followed by type 4 (25%) and type 1 (25%) [Figure 2].
In HIV cases, routine ZN staining detected 4 (25%) cases of AFB positivity and the bleach method detected 14 (87.5%) cases of AFB positivity.
Though the microbiologists are familiar with concentration techniques for an improved yield of mycobacteria for microscopic demonstration and culture, it is a less explored arena for cytologists. Cytological diagnosis based on morphology and confirmation by a positive ZN stain for AFB is a time-tested method for the diagnosis of tuberculous lymphadenitis. The routine ZN stain has low sensitivity and in this study it was 12.5%, and similar results were obtained by several other authors. However, the detection rate is much better with the modified bleach method and in the present study it was 60.7%. Auramine-rhodamine stain is considered better than routine ZN stain in sensitivity, but is less specific. Majority of the positive cases showed high AFB positivity that was making them easily visible and detectable, and the bacilli were seen in clumps in a thin background, making the screening process easier, faster and less strenuous on the eye [Figure 3]. AFB morphology was observed to be better preserved in the bleach method, thus reducing the time required for examination of the slides for detection of AFB.
Laboratory acquired infection (LAI) with Mycobacterium tuberculosis is 3–9 times greater than for the general population. Yearly incidence of tuberculosis among laboratory staff is around 0.3 per 1000 people. Use of the bleach method would definitely lower the risk of laboratory infection. Because NaOCl kills the mycobacterium, this method cannot be used on samples intended for culture.
The predominant morphological pattern in HIV-positive cases is 6, followed by 4 and 1. Most of them showed necrotizing with suppurative morphology. In HIV-positive individuals, AFB positivity by bleach method in this study was 87.5%, but the routine ZN staining showed only 25%.
An attempt was made to correlate the number of bacilli per high-power field in conventional versus bleach method. The number of bacilli is more in bleach method and in three cases the bacilli were seen in clusters: two were of pattern 4 and one was of 6 [Figure 3d]. More important was the ease with which they were identified because of the clean background. In our observation, bacilli in the bleach method were slightly thicker and longer than the routine ZN smears, probably due to swelling of the bacilli in the liquid.
The percentage of AFB positivity in the present study was marginally lower (60.7%) compared to that reported in previous studies by Gangane et al., and Annam et al. (72% and 63.4%, respectively). The reason probably is more number of pattern 2 cases in the present study.
It is a common belief that due to their lipid coat, mycobacteria remain buoyant during centrifugation. Contrary to this belief, the bleach technique has been able to allow the deposition of bacilli at the bottom of the test tube after centrifugation. Annam et al. explained this improved recovery is probably due to changes in the surface properties of the bacilli (i.e. charge and hydrophobicity) and/or denaturation of the specimen leading to flocculation and subsequent increased sedimentation rate of the AFB. We observed that the yield was good when the duration of bleach was 30 min to 1 h. The yield gradually decreased with prolonged time of bleaching. Hence, it may not be possible to get good results in field studies where one needs to process the material after days. Though it was not tried in this study, one may think of collecting the material in formalin initially and performing bleach dilution subsequently.
The present study validates the literature on the utility of the concentration of AFB by modified bleach method for detection of tuberculous bacilli in lymph node aspirates and is more sensitive than the conventional ZN method. Moreover, the modified bleach method is safe, easy to perform and inexpensive.
Source of Support: Nil
Conflict of Interest: None declared.