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.[11
] 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 . 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.
(a) Routine ZN smear (ZN stain, ×1000); (b, c, d) AFB in bleach smears (ZN stain, ×1000); (d) clumps of bacilli (ZN stain, ×1000). Arrows show tuberculous bacilli
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.[12
] 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 . 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.