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Year : 2021  |  Volume : 12  |  Issue : 2  |  Page : 80-85

Comparative study of conventional Ziehl−Neelsen and bleach concentration method in detecting acid-fast bacilli in fine-needle aspiration material of lymph nodes

1 Department of Pathology, Bankura Sammilani Medical College, Bankura, West Bengal, India
2 Department of Pathology, Burdwan Medical College, Bardhaman, West Bengal, India
3 Department of Pathology, Medical College, Kolkata, West Bengal, India

Date of Submission03-Aug-2021
Date of Acceptance22-Sep-2021
Date of Web Publication28-Feb-2022

Correspondence Address:
Dr. Soma Ghosh
Bahir Sarbomangala Road, Near IIHT Computer Centre and Carnival Marriage Hall, Burdwan - 713 101, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjmsr.mjmsr_32_21

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Background: Tuberculosis (TB), specific infectious disease, affects most of the organs of the human body. Extrapulmonary TB manifests commonly as lymphadenopathy. Emergence of acquired immunodeficiency syndrome warrants the rapid diagnosis to limit its spread. Aim and Objectives: The application of bleach concentration method in detecting tubercle bacilli in the material obtained by fine-needle aspiration (FNA) of lymph nodes, evaluating sensitivity of bleach concentration method over conventional direct smear method in diagnosing tubercular lymphadenitis, detecting bacilli in the background of granuloma formation. Materials and Methods: The prospective, hospital-based, cross-sectional study was carried out in a study population of 78 patients where 15 cases got eliminated following the cytological diagnosis of malignancy and inadequacy of sample. Patients with lymphadenopathy underwent FNA after clinical examination, history taking, and informed consent. The aspirated material was stained with Leishman, H and E, conventional Ziehl−Neelsen (ZN), and bleach concentration method. Data were collected and statistical analysis done. Results: The predominant age group affected was (16–30) years with male preponderance. 44.44% cases were cytomorphologically diagnosed with tuberculous lymphadenitis. Acid-fast Bacilli (AFB) was found in 23 cases by conventional ZN method and 39 cases by bleach concentration method. Sensitivity of bleach method was 92.35% compared to 54.76% in conventional ZN method. Bacilli positivity was more in cases presenting with necrosis than with granuloma alone. Conclusion: Bleach method for detecting AFB is more sensitive than conventional ZN method. It is safe, inexpensive, easy to perform, requires no additional equipment, and safe to handle in laboratory.

Keywords: Aspirate, bleach, lymph node, tuberculosis, Ziehl−Neelsen

How to cite this article:
Mandal P, Ghosh S, Banerjee A, Banerjee U. Comparative study of conventional Ziehl−Neelsen and bleach concentration method in detecting acid-fast bacilli in fine-needle aspiration material of lymph nodes. Muller J Med Sci Res 2021;12:80-5

How to cite this URL:
Mandal P, Ghosh S, Banerjee A, Banerjee U. Comparative study of conventional Ziehl−Neelsen and bleach concentration method in detecting acid-fast bacilli in fine-needle aspiration material of lymph nodes. Muller J Med Sci Res [serial online] 2021 [cited 2023 Mar 26];12:80-5. Available from: https://www.mjmsr.net/text.asp?2021/12/2/80/338498

  Introduction Top

Tuberculosis (TB), specific infectious disease is caused by Mycobacterium tuberculosis. Apart from affecting lungs, it can also affect intestine, meninges, bone and joints, lymph node, skin, and other tissues of the body. TB remains a worldwide public health problem despite the fact that the causative organism was discovered more than a 100 years ago.[1] Every day more than 20,000 people become infected with tubercle bacillus, more than 5000 develop the disease and more than 1000 die from it. Emergence of human immunodeficiency virus (HIV) added to the burden of disease.[1],[2]

Extrapulmonary TB most commonly manifests as lymphadenopathy.[3] The clinical parameters for diagnosing TB in lymph nodes are neither specific nor does their absence exclude its involvement.[3] However, the conventional Ziehl−Neelsen (ZN) method for acid-fast bacilli (AFB) plays a key role in diagnosing and monitoring treatment in TB. Its major disadvantage is low sensitivity, ranging from 20% to 43%. India accounts for nearly one-third of the global burden of TB and is the most important public health problem faced in this country.[3],[4]

It has become a major barrier to socioeconomic development. Extrapulmonary TB is on the increase world over, which is due to HIV infection epidemic. In India, in general out patients, 10%–20% of new TB cases may be extra pulmonary, while it could be 50% among HIV-positive patients. In India and other developing countries, TB lymphadenitis (TBLN) continues to be the most common extrapulmonary form.[4] Many patients present with peripheral lymphadenitis, which may remain asymptomatic for longer periods and produce disease only when the host resistance is lowered.[4],[5]

The utility of fine-needle aspiration cytology (FNAC) in diagnosing TBLN has been highlighted in a number of studies during the last two decades. In developing countries such as India, the only practical available method for diagnosing extrapulmonary TB is direct smear microscopy for tubercle bacilli in sample from the lesion.[4],[6] Minimum number of tubercle bacilli necessary to produce a positive smear result has been estimated to be minimum of 5000–10,000 per ml.[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 and cannot be used routinely. Serologic techniques have the disadvantage of lack of sensitivity and specificity.[4],[6],[7]

Newer molecular techniques such as polymerase chain reaction, although rapid, are costly for routine use.[6],[7] Microscopy has many advantages when it comes to speed and feasibility, and on improvement of sensitivity, it can become an even more valuable tool for TB control programs worldwide.[4],[6],[7] There are various concentration methods for improving the sensitivity of detecting tubercle bacilli in specimen.[6],[7] Bleach concentration method for the detection of tubercle bacilli has been recently described for sputum, extrapulmonary specimens, and studies have shown improved the detection of bacilli.[5] Hence, the present study was performed to emphasise the role of bleach concentration method over conventional direct smear microscopy for detecting tubercle bacilli in material by fine-needle aspiration (FNA) of lymph nodes.

Aim and objectives

The application of bleach concentration method in detecting tubercle bacilli in material obtained by FNA of lymph nodes, evaluating sensitivity of bleach concentration method over conventional direct smear method in diagnosing tubercular lymphadenitis, detecting bacilli in the background of granuloma formation.

  Materials and Methods Top

The study was conducted in the department of pathology in collaboration with the department of chest medicine of a teaching institute in West Bengal. The study commenced from May 2015 to April 2016 following permission taken from the Institutional Ethics Committee. The study population comprised of 78 patients of all age groups with clinical suspicion of tubercular lymphadenitis. The exclusion criteria were cytological diagnosis other than TB of lymph node. The study design was prospective, hospital-based cross-sectional one. The study tools used were predesigned and pretested pro forma for data collection, 10 ml syringe with 22G needle, slides, Leishman stain, Z-N staining items, 5% sodium hypochlorite (NaOCl) solution, centrifuge, light microscope.

Patients with lymphadenopathy were subjected to brief clinical examination. Data regarding age, sex, duration, description of swelling regarding its site, number, and size were documented for each patient. An informed written consent was taken from patient after explaining the procedure. FNA was performed maintaining strict aseptic measures. Aspiration was carried out allowing the patient in the lying position which exposed the target area. The type of aspirated material was observed.

The collected material was processed for cytological examination on direct and alcohol-fixed smears by staining with H and E (H and E) and Leishman stain. Air dried smears were stained with Z-N stain and examined for the presence of AFB under oil immersion (×100) lens using the light microscope. Alcohol-fixed slides with smeared side up were placed on the staining rack serially at interval of 1 cm. Slides were covered individually with filtered Ziehl's carbol fuchsin working solution followed by heating from underneath with the flame of an alcohol lamp until vapor starts to rise. Staining solution was never allowed to boil or dry. Slides kept covered with staining solution for 5 min by re-flaming as needed allowed by gentle rinsing to remove excess carbol fuchsin.[4]

Slides then covered with 20% sulfuric acid and allowed to stand for 3 min followed by gentle washing with water. Counterstaining with 0.3% methylene blue continued for a minute and then rinsed with water and allowed to dry. Aspirate from the needle was mixed with 2 ml of 5% NaOCl for the bleach concentration technique. The mixture was incubated for 15 min at room temperature with intermittent mixing. An equal volume of distilled water was added and mixed thoroughly followed by centrifugation at 3000 rpm for 15 min. Smear prepared using one drop of the sediment on discarding the supernatant, air dried, heat fixed and stained by Z-N staining method. A Z-N stained smear made from centrifuged deposit of 2 ml of distilled water was treated as control. Smears were scanned under oil immersion (×100) for detecting AFB.

Statistical analysis was performed using SPSS 20 (Version 20, SPSS INC., Chicago, IL, USA) where Chi-square (χ2) test showed significance. Calculation was performed for sensitivity, specificity, and positive predictive value.

  Results Top

In a study population of 78 cases, 15 cases got eliminated on the basis of cytological diagnosis. Malignancy was diagnosed in 12 aspirates and inadequate material found in three. Eventually, 63 clinically suspected tubercular lymphadenitis cases were evaluated. Most patients (n = 26, 41.26%) were in the age group of 16–30 years. The youngest patient was of 1 year and oldest one was 70 years old. A male preponderance was noted in this study (52.39%) with a male-to-female ratio of 1:0.90. Lymphadenopathy with a history of <3 months was elicited in 38 patients (60.31%). Cervical lymphadenopathy was found in 42 patients (66.67%). Fifty-three patients (84.12%) presented with lymph nodes with size of 1–2 cm. Multiple matted lymph nodes found in 32 patients (50.80%). Aspirate obtained by FNA was gray white and granular in 36 cases (57.14%). Cytomorphological diagnosis of tuberculous lymphadenitis found in 28 cases (44.45%) [Table 1].
Table 1: Clinical profile with cytological diagnosis in the study population

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In the present study, 23 (36.50%) cases were AFB positive by conventional Z-N method and 39 (61.90%) by bleach concentration method; with varying grades of positivity [Table 2].
Table 2: Comparison of acid-fast bacilli grading by conventional Ziehl-Neelsen and bleach method

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The comparison between cytomorphological diagnosis and conventional Z-N method showed statistical significance where Chi-square test, χ2 = 16.931, degree of freedom (df) =2, P = 0.000. The comparison between cytomorphological diagnosis and bleach method also showed statistical significance where, Chi-square test, χ2 = 19.825, degree of freedom (df) =2, P = 0.000. Even, there was statistically significant correlation (Chi-square test, χ2 = 22.292, df = 1, P = 0.000) between the results of bleach method and conventional method in detecting AFB [Table 3].
Table 3: Ziehl-Neelsen and bleach method in various lymphadenopathies

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One of six cases showed AFB positivity presenting only with granuloma, 10 of 14 cases showed AFB positivity where granuloma was coupled with necrosis. Six of eight cases showed AFB positivity in smears with only necrosis [Table 4].
Table 4: Comparison between smears showing granuloma/necrosis and bacilli positivity

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The sensitivity for detecting AFB in aspiration cytology smears increased from 54.76% by conventional ZN staining to 92.35% by modified bleach method [Table 5].
Table 5: Evaluation of bleach method and conventional method

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  Discussion Top

The increasing number of TB cases in recent years and the emergence of strains with multidrug resistance present a public health problem that requires rapid intervention.[8] The emergence of acquired immunodeficiency syndrome has exacerbated the state. Rapid diagnosis of the disease considered mandatory to confine it.[8],[9] Widespread manifestation of the disease makes the diagnosis easier compared to localized extrapulmonary involvement.[9]

Early diagnosis and initiating optimal treatment would not only enable cure but will curb the transmission of infection and disease to others in the community.[10] The diagnostic dilemma with extrapulmonary TB in developing countries can be countered by FNAC which can ensure prompt treatment, thus reducing morbidity and mortality.[10],[11] ZN stain for AFB confirms the diagnosis in the presence of inflammatory exudates.[11]

AFB positivity is higher in untreated, wide dissemination, immunecompromised, and HIV-positive patients.[12] The use of bleach can be traced back to 1909, where NaOCl (bleach) was used as mucolytic before centrifuging sputum samples and further processed by direct microscopy after the Z-N staining method. Bleach method has undergone several modifications, diversifications and has recently received more attention with the advent of HIV epidemic. Bleach is cheap, readily available and improves sensitivity of the direct microscopy method considerably.[12],[13] It is also an effective disinfectant, and therefore, helps in reducing infection risk in laboratory personnel.[13]

The bleach method has a special advantage in overburdened control programs, where the technicians cannot afford to spend the required time for the examination of one slide.[14] Sensitivity by the bleach method gets improved to 70% compared to 55% by conventional one.[15],[16] The technical method is advantageous in control programs where NaOCl is affordable and available. High density of bacilli per microscopic field and clear background imparts improved sensitivity in bleach method.[16]

The discrepancies between cytomorphological diagnosis and bleach method in the present study were found in 16 samples where reactive lymphadenitis and acute suppurative lymphadenitis found in four samples each. These samples were positive for AFB by bleach method. The presence of bacilli in smears of reactive lymphadenitis can be due to scattered epithelioid cells among polymorphous lymphoid population.

Among the four samples diagnosed with suppurative lymphadenitis but positive for AFB by bleach method, it could be due to failure in recognizing bacilli among the necrotic debris. Khubnani and Munjal in their study included 18 aspirates from body fluids, 18 from abscesses, 17 from lymph nodes, and two from skin scrapings in 55 cases of extrapulmonary TB. They reported 43.36% cases of TB on cytology where 21.8% cases were positive for AFB by conventional Z-N staining and 70.90% cases by the bleach method.[17] Gangane et al. studied 100 cases of TBLN and found AFB positivity in 72% of cases by the bleach concentration method. AFB positivity grade was much higher than with routine Z-N staining making bacilli easily visible with shorter screening time [Table 6].[18]
Table 6: Comparison of acid-fast bacilli positivity in different studies by conventional and bleach method

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Annam et al. studied 93 cases of lymphadenopathy, 33.33% were positive for AFB by conventional Z-N method, and 63.44% on bleach method. They concluded bleach method as simple and inexpensive.[19] Chandrasekhar and Prayaga studied 112 cases of lymph node aspirate, among which TB was diagnosed in 60.7% on cytology, conventional Z-N staining for AFB was positive in 12.5%, and bleach method for AFB was positive in 63.44%.[20] Patel et al. studied 115 cases of lymph node aspirate, among which 59.13% cases were suggestive of TB on cytology, 29.41% cases positive for AFB by conventional Z-N staining, and 66.18% cases positive for AFB by the bleach method [Table 6].[21]

Gunja and Chandra studied 200 cases of lymph node aspirate, among which 52% cases were suggestive of TB on cytology, 35.5% cases positive for AFB by Z-N staining, and 68% were positive by modified bleach methods.[22] In the present study of 63 cases of lymph node aspirate, 44.44% cases were suggestive of TB on cytology, 36.50% cases positive for AFB by conventional Z-N staining, and 61.90% cases positive for AFB by bleach method. The study is limited by low size of sample with short duration.

Lakhey et al. studied 122 cases of tubercular lymphadenitis and found granuloma alone in 31 cases (25.5%) out of which ZN positive was in five cases (16.1%), granuloma with necrosis in 57 cases (46.7%) of which ZN positive was in 38 cases (66.6%), necrosis alone found in 34 cases (27.8%) where ZN positive was in 28 cases (82.3%).[23] In the present study of 28 cases of TBLN, one of six cases showed positivity by ZN stain with only granuloma (16.66%), 10 of 14 showed positivity by ZN stain with granuloma and necrosis (71.42%), six of eight cases showed positivity by ZN stain with necrosis alone (75%).

Here, the liquefaction of the aspirated specimen with NaOCl followed by centrifugation significantly increased the yield of AFB. This technique is advantageous in smear negative AFB cases. NaOCl treatment for improved recovery of AFB is attributed to the changes in charge on surface and hydrophobic properties of AFB. Denaturation of the specimen leads to flocculation and subsequent increased sedimentation rate of AFB. Furthermore, the increased smear positivity by bleach method is attributable to more density of bacilli per microscopic field and a clear background for microscopy.

Subsequently, preparation of samples by the bleach method reduces time required for examination. Smear examination by bleach method does not discriminate between tubercle bacilli and other mycobacteria. Majority of population in developing countries with AFB has TB or other mycobacteria which are usually not present in sufficient concentration to be detected by direct microscopy. Multi-drug resistant TB has become a major concern for infection among laboratory personnel and bleach method helps in limiting such risk. NaOCl kills the mycobacterium and cannot be applied for culture but strongly recommended for direct microscopy. Implementing bleach method is of utmost help for diagnosing TB by the microscopic detection of bacilli eventually beneficial in early and effective treatment. AFB is more populous in smears presenting with necrosis or granulomas compounded with necrosis.

  Conclusion Top

Early identification and isolation of TB patients are of utmost importance in minimizing the risk of further epidemic spread. Combination of fine FNAC with acid fast staining is highly valuable for routine diagnosis of TB. Bleach method for detecting tubercle bacilli in lymph node aspirate is more sensitive than the conventional Z-N method. It is safe, inexpensive, easy to perform, and limits risk of laboratory-acquired infections with no need of additional equipment.

Declaration of patient consent

We do certify that we have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

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Conflicts of interest

There are no conflicts of interest.

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