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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 16  |  Issue : 2  |  Page : 349-353

Comparative study of conventional ziehl–neelsen method and modified bleach concentration method in detection of tubercle bacilli in fine-needle aspiration material of lymph nodes


Department of Pathology, Jawaharlal Nehru Medical College, Datta Meghe Institute of Medical Sciences (Deemed to be University), Sawangi (M), Wardha, Maharashtra, India

Date of Submission17-Apr-2020
Date of Decision08-Oct-2020
Date of Acceptance10-Jan-2021
Date of Web Publication18-Oct-2021

Correspondence Address:
Dr. Ankita Laddha
Shalinata PG Girlsf Hostel, JNMC Campus, Sawangi (Meghe), Wardha, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_133_20

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  Abstract 


Introduction: Tuberculous lymphadenitis is seen in nearly 35% of extrapulmonary tuberculosis. Early and accurate detection of active cases remains an important objective for appropriate treatment and reduction in the spread of the disease. Microscopy has many advantages when it comes to speed feasibility, and if its sensitivity could be improved. Liquefaction of specimens by sodium hypochlorite (NaOCl, bleach) and concentration of bacilli through centrifugation significantly increases the sensitivity of direct microscopy. Thus, the bleach concentration method has been recently described for sputum and other extra-pulmonary specimen. Aim: The aim of study is to evaluate the role of bleach concentration method in FNAC lymph nodes over conventional Ziehl–Neelsen (ZN) direct smear microscopy. Methods and Material: The prospective, cross-sectional and observational study carried out for a period of one year from December 2017 to November 2018. The study included 50 cases suspected clinically of having TB with lymphadenopathy referred for FNAC to the division of cytopathology in department of Patholology, All the aspirates by FNAC were processed for direct microscopy using conventional ZN staining and routine cytology and compared with the findings of the bleach method. Of these 50 cases 5 cases were eliminated from the study. Results: Of total 45 cases, 14 cases were positive by conventional ZN method and 29 cases by bleach concentration method. AFB positivity by bleach concentration method was more in comparison with the routine ZN stain. Conclusion: The study could establish AFB positivity in 64.44% of cases with the bleach method. This detection rate is far better than routine ZN staining.

Keywords: Acid fast bacilli positivity, bleach concentration method, conventional Ziehl–Neelsen method, tuberculous lymphadenitis


How to cite this article:
Laddha A, Bhake A, Kediya A, Vagha S. Comparative study of conventional ziehl–neelsen method and modified bleach concentration method in detection of tubercle bacilli in fine-needle aspiration material of lymph nodes. J Datta Meghe Inst Med Sci Univ 2021;16:349-53

How to cite this URL:
Laddha A, Bhake A, Kediya A, Vagha S. Comparative study of conventional ziehl–neelsen method and modified bleach concentration method in detection of tubercle bacilli in fine-needle aspiration material of lymph nodes. J Datta Meghe Inst Med Sci Univ [serial online] 2021 [cited 2021 Dec 9];16:349-53. Available from: http://www.journaldmims.com/text.asp?2021/16/2/349/328447




  Introduction Top


Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis and continues to be a major health problem in developing countries. While pulmonary TB is the most widespread presentation, extra-pulmonary TB (EPTB: Affecting outside the lung) is also an important clinical problem. Lymphadenopathy is the most common presentation of EPTB. Tuberculous lymphadenitis can be presumptively diagnosed morphologically on fine-needle aspiration cytology (FNAC) of the lymph node.[1],[2] Conventional Ziehl–Neelsen (ZN) method for acid-fast bacilli (AFB) plays a key role in the diagnosis and monitoring of TB treatment.[3],[4] The bleach concentration method has been recently described for sputum and other extra-pulmonary specimen.[5]


  Materials and Methods Top


It was a prospective, cross-sectional, and observational study conducted from December 2017 to November 2018 over a period of 1 year. An informed consent was taken from all the patients who were included in the study.

Inclusion criteria

The patients of suspected clinically of having TB with lymphadenopathy referred for FNAC to the division of cytopathology in the department of pathology were included in the study.

Exclusion criteria

The patient on treatment for TB within the previous 3 months or initiation of TB treatment before sampling was performed.

The study was carried out on 50 patients with clinically suspected tuberculous lymphadenopathy, referred to the department of cytopathology. They were subjected to a brief clinical examination. Data regarding age, gender, duration, and description of swelling-like site was documented for each patient. FNAC was performed with a 23-gauge needle and a 10-ml disposable syringe.

Of these 50 cases, 5 cases were eliminated from the study because 4 aspirates were diagnosed as malignancy and 1 aspirate was inadequate. Therefore, total sample size was 45 cases. All the aspirates by FNAC were processed for direct microscopy using conventional ZN staining and routine cytology and compared with the findings of the bleach method.

Conventional cytology technique:[6] For cytological examination, approximately 5–6 smears were prepared on clean glass slides. Smears were prepared directly and wet-fixed smears (in absolute alcohol) were stained by hematoxylin and eosin and Papanicolaou (Pap) stains while air-dried smears were stained with May–Grunwald Giemsa and routine ZN stains, respectively. ZN stained smears were examined for the presence of tubercle bacilli. Other smears were studied for cytomorphological evidence of TB.

Bleach concentration technique:[6] The residual aspirated material in the needle was flushed out and subjected to liquefaction with 5% sodium hypochlorite (NaOCl, bleach) and stained with the routine ZN method.

Tuberculous lymphadenitis had been diagnosed based on the cytomorphological features of FNA smears. Cytomorphological criteria defining cytological diagnosis of reactive, suppurative, and granulomatous lymphadenitis were as follows:

  1. Reactive lymphadenitis revealed mixed population of lymphoid cells in varying proportions along with scattered histiocytes with intracytoplasmic nuclear debris (tingible body macrophages)
  2. Suppurative lymphadenitis revealed lymphoid cells along with numerous intact and degenerated polymorphs against a background of necrotic debris
  3. Granulomatous lymphadenitis showed epithelioid cell granulomas with or without the presence of Langhans' multinucleated giant cells and caseous necrosis.


Cytological patterns in cases diagnosed as tuberculous lymphadenitis include:

  1. Epithelioid granuloma with extensive necrosis
  2. Epithelioid cell granulomas only
  3. Necrosis only [Table 1],[Table 2],[Table 3],[Table 4],[Table 5],[Table 6],[Table 7],[Table 8],[Table 9],[Table 10],[Table 11],[Table 12].[6]
Table 1: Distribution of cases according to age at diagnosis

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Table 2: Distribution of cases according to gender

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Table 3: Distribution of cases according to the duration of lymphadenopathy

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Table 4: Distribution of cases according to group of lymph node involvement

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Table 5: Distribution of cases according to size of lymph node

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Table 6: Distribution of cases according to gross description of lymph nodes

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Table 7: Distribution of cases according to cytomorphological diagnosis

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Table 8: Distribution of cases according to conventional Ziehl- Neelsen Method for AFB

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Table 9: Distribution of cases according to bleach concentration method

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Table 10: Correlation of cytomorphological diagnosis with the bleach method and the Conventional Ziehl- Neelsen method

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Table 11: Comparison of conventional Ziehl- Neelsen method with Bleach method for detection of acid fast bacilli

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Table 12: Comparison of AFB positivity in different studies by conventional Ziehl- Neelsen and Bleach concentration method

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  Results Top


The present study was undertaken to emphasize the role of bleach concentration method over conventional direct smear microscopy for detection of tubercle bacilli in fine-needle aspirate material of lymph node. This study was carried out on 50 patients with clinically suspected tuberculous lymphadenopathy, referred to the department of cytopathology.

Of these 50 cases, 5 cases were eliminated from the study because 4 aspirates were diagnosed as malignancy and 1 aspirate was inadequate and 45 clinically suspected tuberculous lymphadenopathy cases evaluated and the results were analyzed as follow:

In the present study, newly recommended bleach concentration method was utilized for detection of AFB in the diagnosis of tubercular lymphadenitis. Aspirates of all 45 cases suspected of tubercular lymphadenitis were subjected for bleach concentration method and smears stained by ZN method and searched for acid-fast bacilli.

In the present study, total 29 cases were AFB positive (64.44%) and 16 cases were AFB negative by bleach concentration method.

Of total 45 cases, 14 cases were positive both on conventional Z-N method and bleach concentration method. By bleach concentration method, 15 additional cases showed positivity that was not revealed by the conventional ZN method. Thus, a total of 29 cases were positive for AFB by the bleach concentration method. None of the cases diagnosed positive on routine ZN stain was missed by the bleach concentration method. AFB positivity by bleach concentration method was more in comparison with the routine ZN stain [Figure 1] and [Figure 2].
Figure 1: Acid-fast bacilli positive by Ziehl–Neelsen technique (×100)

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Figure 2: Acid-fast bacilli positive by Ziehl–Neelsen technique (×100)

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  Discussion Top


Tuberculous lymphadenitis is the most common type of EPTB. The diagnosis of TB is easy and simple when the disease is elaborate or disseminated, however, localized involvement of extra-pulmonary organ or tissue may at times pose a diagnostic problem. The clinical parameters for the diagnosis of TB in lymph nodes are neither specific nor do their absence exclude tubercular involvement. Early diagnosis of TB and initiating optimal treatment would not only enable a cure of an individual patient but will also curb the transmission of infection and disease to others in the community.

Diagnostic modalities must also be tailored to the needs of the population and epidemiology of TB in that region. These include improved microscopy, usage of liquid culture for childhood and EPTB, chemical and physical detection of mycobacterial antigens in paucibacillary condition, antigen capture, antibody detection, cellular immune recognition, nucleic acid amplification, and phage assay.[6]

Various techniques other than the conventional ZN staining and bleach concentration method for demonstrating AFB are Pap stain, auramine rhodamine staining (ARS), autofluorescence stain, fluoresceindiacetate (FDA) vital staining, light emitting diodes (LED) microscopy, and polymerase chain reaction (PCR) technique.

Mycobacterial culture is the reference method and is time consuming. It requires specialized safety procedures in laboratories. Serological techniques lack sensitivity and specificity. PCR, although rapid, is costly to be routinely used in developing countries where TB is prevalent.[6]

Pap, ARS, FDA, and LED techniques require a fluorescent microscope. Availability of fluorescent microscopes in laboratories with resourcelimited settings is difficult. Advantage of fluorescent microscopy is that it can be used at lower magnifications and time required is less to examine much larger area per unit time. Pap stain avoids the use of toxic or carcinogenic substances such as phenol and rhodamine used in ARS method.[6] ZN method for AFB bacilli plays a key role in the diagnosis as well as in the monitoring of TB treatment.[7],[8]

Bleach concentration method is one of the safest methods for improving the sensitivity of direct microscopy for the detection of AFB. A few previous studies have shown that liquefaction of sputum by NaOCl and concentration of bacilli through centrifugation significantly increases the sensitivity of direct microscopy.[9],[10] In the late 1940s, sputum liquefaction with NaOCl followed by concentration using centrifugation before acidfast staining was implemented to improve the smear positivity for the detection of AFB. This method was slightly modified and applied in lymph node aspirates.

The present study revealed discordance between cytomorphological diagnoses and AFB positivity by bleach method in four cases. Two specimens were reactive lymphadenitis and two specimens were acute suppurative lymphadenitis; however, these were positive for AFB by the bleach method. The possible explanation for the diagnosis of reactive lymphadenitis on cytology but positive for AFB by the bleach method in cases may be due to the loss of scattered epithelioid cells among the polymorphous population of lymphoid cells. Among the two specimens diagnosed as suppurative lymphadenitis, these were positive for AFB by the bleach method, the likely reason could be loss of the bacilli amidst the necrotic debris.

Khubnani et al.[5] studied 55 cases of EPTB, which included 18 aspirates from body fluids, 18 from abscesses drained from various body sites, 17 from lymph nodes, and 2 from skin scrapings. It was found that an overall 43.36% cases were suggestive of TB on cytology, 21.8% cases positive for AFB by conventional ZN staining, and 70.90% cases positive for AFB by the bleach method.

Gangane et al.[11] studied 100 cases of TB lymphadenitis and concluded that bleach concentration method demonstrated AFB positivity in 72% of the cases. AFB positivity grade was significantly higher than with routine ZN staining making bacilli easily visible with shorter screening time. The bleach method was inexpensive, easily performed, and more sensitive and safer than the routine ZN staining.[7]

Annam et al.[12] studied 93 cases of lymphadenopathy. Among 93 aspirates, 33.33% were positive for AFB in conventional ZN method and the smear positivity increased to 63.44% on bleach method. They concluded that the bleach method was simple and inexpensive.[12]

Chandrasekhar et al.[13] studied 112 cases of lymphadenopathy. Among 112 aspirates, 12.5% were positive for AFB in conventional ZN method and the smear positivity increased to 60.7% on bleach method.

Krishna et al.[6] studied 75 cases of suspected tubercular lymphadenopathy and found that 20% were positive for AFB in conventional ZN method and the smear positivity increased to 65.33% on bleach concentration method.

The present study demonstrated that liquefaction of the aspirated specimen with NaOCl followed by centrifugation significantly increases the yield of AFB. This finding is of considerable interest in developing countries where smearnegative AFB has become increasingly common. The improved recovery of AFB after treatment with NaOCl might be due to changes in the surface properties of the AFB (i.e., charge and hydrophobicity) and/or denaturation of the specimen, leading to flocculation and subsequent increased sedimentation rate of the AFB. Moreover, the increased smear positivity by the bleach method is attributable to the higher density of bacilli per microscopic field obtained by the method and reduction of debris, leaving a clear field for microscopy. Thus, the preparation of samples by the bleach method reduces the time required for slide examination to detect AFB.

Acidfast smear examination by the bleach method does not discriminate between tubercle bacilli and other mycobacteria. However, this is not a major problem in developing countries as majority of patients with AFB suffer from TB and other mycobacteria are usually not present in sufficient concentration to be detected by direct microscopy. Mycobacteria have a low specific gravity and may remain buoyant during centrifugation.

With the occurrence of multidrugresistant TB, the risk of laboratory infection has become a major concern. The use of the bleach method would definitely lower the risk of laboratory infection. Because NaOCl (bleach) kills the mycobacterium, this method cannot be used on samples intended for culture, however, the method is strongly recommended for all laboratories that perform direct microscopy only.

In the present study, a centrifugation at 3000 rpm for 15 min yielded increased recovery of mycobacteria. Previous studies done by various authors and the present study revealed that the bleach method for AFB detection is simple, safe, costeffective, and has a high case detection rate. The results would be more wellorganized if concentration by bleach solution, Recycled Cellulose Fiber (RCF), and bleach treatment is as per the time schedule and is comparable. The application of the bleach method clearly improves microscopic detection and can be a useful influence to routine cytology. This would be of advantage to the patients in receiving early and effective treatment.


  Conclusion Top


The morphologic spectrum in tuberculous lymphadenopathy varies widely depending on the stage of the immunity of the host. The presence of epithelioid cells is the first step in diagnosis of tuberculous lymphadenopathy in countries like India where TB continues to be the most common cause of lymphadenopathy compared to other non-TB causes of granuloma formation.

There are problems in arriving at an absolute diagnosis in certain cases of tuberculous lymphadenitis when the aspirate shows polymorphous picture with occasional epithelioid cells and absence of typical Langhans giant cell or caseous necrosis, making it necessary for a definitive diagnosis. In such cases, routine ZN staining shows low sensitivity because it rarely detects AFB in aspirates. However, in the present study, we could establish AFB positivity in 64.44% of cases with the bleach method. This detection rate is far better than routine ZN staining.

It was also observed that, by routine ZN staining, most of the aspirates had scant AFB positivity and searching for them was a tedious, timeconsuming exercise compared to the bleach method. By the bleach method, in a majority of AFB positive cases, AFB was easily visible and detectable. AFB morphology was observed to be better preserved in the bleach method.

Thus, the bleach method for detection of tubercle bacilli in lymph node aspirate is more useful than the conventional ZN method. Moreover, the bleach method is safe, inexpensive, and easy to perform requiring no additional equipment.

Acknowledgment

The authors gratefully acknowledged the dedicated efforts and timely help of the investigators, coordinators, and volunteer patients who were participated in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Dandapat MC, Mishra BM, Dash SP, Kar PK. Peripheral lymph node tuberculosis: A review of 80 cases. Br J Surg 1990;77:911-2.  Back to cited text no. 1
    
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Lau SK, Kwan S, Lee J, Wei WI. Source of tubercle bacilli in cervical lymph nodes: A prospective study. J Laryngol Otol 1991;105:558-61.  Back to cited text no. 2
    
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Daniel TM. Rapid diagnosis of tuberculosis: Laboratory techniques applicable in developing countries. Rev Infect Dis 1989;11 Suppl 2:S471-8.  Back to cited text no. 3
    
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Balows A, Hausler WJ, Hermann KL, Shadomy HJ. Manual of Clinical Microbiology. 5th ed. Washington, DC: American Society for Microbiology; 1991. p. 308-11.  Back to cited text no. 4
    
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Khubnani H, Munjal K. Application of bleach method in diagnosis of extra-pulmonary tuberculosis. Indian J Pathol Microbiol 2005;48:546-50.  Back to cited text no. 5
    
6.
Krishna M, Gole SG. Conventional ZN and Modified bleach method comparison in TB. J Cytol 2017;34:188-92.  Back to cited text no. 6
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Savić B, Sjöbring U, Alugupalli S, Larsson L, Miörner H. Evaluation of polymerase chain reaction, tuberculostearic acid analysis, and direct microscopy for the detection of Mycobacterium tuberculosis in sputum. J Infect Dis 1992;166:1177-80.  Back to cited text no. 7
    
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Friede T. Tuberculosis is Case Detection, Treatment, and Monitoring Questions and Answer. 2nd ed. Geneva: WHO; 2004. p. 14-20.  Back to cited text no. 8
    
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Wilkinson D, Sturm AW. Diagnosing tuberculosis in a resource-poor setting: The value of sputum concentration. Trans R Soc Trop Med Hyg 1997;91:420-1.  Back to cited text no. 9
    
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Habeenzu C, Lubasi D, Fleming AF. 'Improved sensitivity of direct microscopy for detection of acid-fast bacilli in sputum in developing countries. Trans R Soc Trop Med Hyg 1998;92:415-6.  Back to cited text no. 10
    
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Gangane N, Anshu A, Singh R. Role of modified Bleach method in staining of acid-fast bacilli in lymph node aspiration. Acta Cytol 2008;52:325-8.  Back to cited text no. 11
    
12.
Annam V, Karigoudar MH, Yelikar BR. Improved microscopical detection of acid fast bacilli by the modified bleach method in lymph node aspirates. Indian J Pathol Microbiol 2009;52:349-52.  Back to cited text no. 12
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Chandrasekhar B, Prayaga AK. Utility of concentration method by modified bleach technique for the demonstration of acid-fast bacilli in the diagnosis of tuberculous lymphadenopathy. J Cytol 2012;29:165-8.  Back to cited text no. 13
[PUBMED]  [Full text]  


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12]



 

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