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Performance of fine needle aspiration cytology and Ziehl-Neelsen staining technique in the diagnosis of tuberculosis lymphadenitis

BMC Infectious Diseases volume 24, Article number: 633 (2024) Cite this article

Abstract

Introduction

Proper diagnosis of tuberculosis (TB) lymphadenitis is critical for its treatment and prevention. Fine needle aspirate cytology (FNAC) is the mainstay method for the diagnosis of TB lymphadenitis in Ethiopia; however, the performance of FNAC has not been evaluated in the Eastern Region of Ethiopia. This study aimed to evaluate the performance of FNAC and Ziehl-Neelsen (ZN) staining compared with that of GeneXpert for the diagnosis of TB lymphadenitis.

Methods

Fine needle aspiration (FNA) specimens collected from 291 patients suspected of having TB lymphadenitis were examined using FNAC, ZN, and GeneXpert to diagnose TB lymphadenitis. Gene-Xpert was considered the reference standard method for comparison. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa coefficient were determined using SPSS version 25.

Results

The sensitivity, specificity, PPV, and NPV of ZN for diagnosing TB lymphadenitis were 73.2%, 97.4%, 96.2%, and 80.1% respectively. There was poor agreement between ZN and GeneXpert (Kappa=-0.253). The sensitivity, specificity, PPV, and NPV of FNAC were 83.3%, 94.8%, 93.5%, and 86.3% respectively. There was moderate agreement between the FNAC and GeneXpert (Kappa = 0.785).

Conclusion

The fine needle aspiration cytology (FNAC) is a more sensitive test for the diagnosis of TB lymphadenitis than ZN. The FNAC showed a moderate agreement with the GeneXpert assay. This study recommends the FNA GeneXpert MTB/RIF test in preference to FNAC for the diagnosis of TB lymphadenitis to avoid a missed diagnosis of smear-negative TB lymphadenitis.

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Introduction

Tuberculous lymphadenitis (TBL) is a chronic specific granulomatous inflammation that causes necrosis in lymph nodes [1]. It is often caused by reactivation of latent infection and is the most common manifestation of extrapulmonary tuberculosis (EPTB) [2, 3]. One-fourth of the world’s population is latently infected with Mycobacterium tuberculosis [4]. The World Health Organization (WHO) 2022 report shows that there were 10.6 million TB cases and 1.6 million deaths in 2021, compared to 10.1 million cases and 1.5 million deaths in 2020, with a 3.6% increase in the incidence rate [5].

Extrapulmonary TB accounts for 15–20% of all TB cases and accounts for 50% of HIV-confirmed cases [6]. TB lymphadenitis is observed in approximately 35% of EPTB patients [7]. Pulmonary TB and TB lymphadenitis are the most common forms of TB in the world [8]. In Ethiopia, approximately one-third of TB cases are attributed to TB lymphadenitis [9].

Tuberculous lymphadenitis is not part of the global TB response strategy because of its minor role in TB transmission [10]. However, there is evidence that TB lymphadenitis has a future impact on global TB control because of its ability to reactivate TB, its obscure location, its paucibacillary nature and its ability to be diagnosed at an advanced stage of the disease when complications are present [6, 11]. Therefore, proper diagnosis of TB lymphadenitis is critical for its treatment and prevention.

The WHO has endorsed the GeneXpert MTB/RIF assay as the fastest and most sensitive test compared to conventional methods, with greater feasibility for point-of-care implementation due to minimal infrastructure requirements [12]. Also in 2017, WHO confirmed the sensitivity similarity of the next-generation Xpert MTB/RIF ultra assay to solid culture with an improved limit of detection [13].

The microscopic diagnosis of pulmonary TB is essential in developing countries because it is inexpensive, rapid and sensitive, but the sensitivity is limited to 20–43% for EPTB patients [14]. FNAC is an important diagnostic method for EPTB [15]. In Ethiopia, FNAC is the mainstay method for the diagnosis of TB lymphadenitis; however, the performance of FNAC has not been evaluated in the Eastern Region of Ethiopia. This study aimed to evaluate the performance of FNAC and Ziehl-Neelsen (ZN) staining technique in combination with GeneXpert for the diagnosis of TB lymphadenitis at Adama Hospital Medical College (AHMC), Adama, Ethiopia.

Materials and methods

Study area and design

A hospital-based cross-sectional study was conducted among 291 patients suspected of having TB lymphadenitis at AHMC, Adama, Ethiopia, from May to August 2022. The city is located about 99 km due Southeast of Addis Ababa, the capital city of Ethiopia. It is located at latitude of 8° 54’ north and a longitude of 39° 27’ east. AHMC serves as a referral hospital for the people residing in the East Shewa Zone of the Oromia Regional State and adjacent regions. Sociodemographic and clinical data was collected using a structured preprepared format [16]. For the diagnosis of TBL, the ZN staining technique, FNAC, and GeneXpert were used. GeneXpert was considered the reference standard to which other methods were compared and the performance was determined accordingly.

Sample collection and processing

Approximately 1.5-3 ml fine needle aspiration (FNA) samples were collected from the enlarged superficial lymph nodes using 22–23 gauge needles. One part of the sample was transferred to a sterile container with normal saline for GeneXpert and the remaining part was smeared on two different slides for cytological and Ziehl-Neelsen staining.

Cytological diagnosis

The air-dried FNA smears were stained with Wright’s stain according to the SOPs [16] and examined microscopically by experienced pathologists. The FNAC shows up the epithelioid granulomas, and the epithelioid granulomas with multinucleated giant cells, caseous necrosis, degenerating inflammatory cells and liquefied necrotic materials were considered cytologically positive for tuberculosis [16, 17].

Ziehl-Neelsen (ZN) staining

ZN staining was performed according to the SOP [16], and the stained smears were examined by microbiologists at AHMC under the oil-immersion objective (100*) of a light microscope. A minimum of 100 fields were scanned for negative results.

GeneXpert MTB/RF assay

The GeneXpert MTB/RIF assay (Cepheid, CA, USA) was performed according to microbiology laboratory SOPs [16]: 2 ml of Gene-Xpert MTB/RIF Specimen Reagent Buffer was added to 1 ml of fine needle aspiration sample using a sterile pipette. The closed sample container was manually vortexed twice for 15 s, allowed to stand at room temperature for 10 min, vortexed after 10 min and allowed to stand for 5 min. Then 2 ml of the inactivated material was transferred to the test cartilage and the cartilage was loaded into the Gene-Xpert device. Finally, the results were interpreted by the Gene-Xpert diagnostic system from the measured fluorescence signals and automatically displayed after 2 h [16].

Data analysis

The data were analyzed using SPSS version 25. The sensitivity, specificity, PPV, NPV, and Kappa coefficient were determined. The agreement between the tests and the reference method were evaluated using the Kappa value. The Kappa values 0-0.2, 0.21–0.39, 0.4–0.59, 0.6–0.79, 0.8–0.9, > 0.9 were interpreted as no agreement, minimal agreement, weak agreement, moderate agreement, strong agreement, and almost perfect agreement respectively [18].

Ethical clearance

The study was approved by the Institutional Review Board of Hawassa University Medical College (reference number: IRB/148/14). The purpose and procedure of the study were explained to the study participants. Data were collected after written informed consent and/or assent was obtained from the study participants or the parents/guardians. Strict confidentiality was maintained throughout the study using only code.

Results

A total of 291 FNAC samples were performed using cytology, ZN and GeneXpert methods. Among the 291 samples, about 138 (47.4%) were positive according to the reference standard method GeneXpert, 123(42.3%) were cytologically suggestive of TB and 105(36.1%) were positive for acid fast bacilli (AFB) in ZN staining microscopy. The sensitivity, specificity, PPV, and NPV of ZN for diagnosing TB lymphadenitis were 73.2%, 97.4%, 96.2%, and 80.1% respectively. There was poor agreement between ZN and GeneXpert (Kappa=-0.253). The sensitivity, specificity, PPV, and NPV of FNAC were 83.3%, 94.8%, 93.5%, and 86.3% respectively. There was moderate agreement between the FNAC and GeneXpert (Kappa = 0.785) (Table 1).

Table 1 Diagnostic performance of fine needle aspiration cytology and Ziehl-Neelsen staining technique compared to the GeneXpert reference standard in the diagnosis of tuberculosis lymphadenitis
Full size table

Discussion

One of the mainstays of TB care and control is accurate and timely diagnosis and effective treatment. In developed countries, a confirmed diagnosis of TB can only be made by culture or by finding a specific DNA sequence of the bacteria in a sputum sample for pulmonary TB and FNAC for extrapulmonary TB. However, in developing countries such as Ethiopia, these tests are not available in all areas of the country. In these countries, cost-effective techniques such as the ZN staining method and, in the case of EPTB, FNAC are very useful methods for detecting tuberculosis.

The sensitivity, specificity, PPV and NPV of ZN staining for the diagnosis of TB lymphadenitis in the present study were 73.2%, 97.4%, 96.2% and 80.1%, respectively. The sensitivity of ZN in the present study was lower than that reported from Tirupati, India (83.3%) [19] and India(91%) [20], while it was higher than that reported from Bangladesh (17.6%) [21] and Ethiopia (22.9%) [22]. However, the specificity of ZN staining in the present study (80.1%) was lower than that reported in Bangladesh (98.4%) [21], Ethiopia (92.4%) [22], Tirupati, India (88.9%) [19], India (90%) [20] and South Africa, 88.9% [23]. The percentage of agreement of ZN staining with GeneXpert was − 0.253 (Kappa test), indicating no agreement.

In the present study, the sensitivity, specificity, PPV and NPV of FNAC for the diagnosis of TB lymphadenitis were 83.3%, 94.8%, 93.5% and 86.3%, respectively. The sensitivity of FNAC in the present study was comparable to that reported Ethiopia (81%) [22] and Bangladesh (79.7%) [21] but lower than that reported in Egypt (90.9%) [24]. However, the specificity of FNAC in the present study (86.3%) was greater than that reported in Bangladesh (48.1%) [21], Ethiopia (50%) [22] and Egypt (67.2%) [24]. The percentage of agreement of FNAC with GeneXpert was 0.785 (Kappa test), indicating moderate agreement, which was similar to the results of a study conducted in India, where the percentage of agreement of FNAC with GeneXpert was 0.4 [25]. The highest sensitivity of TB lymphadenitis in ZN and FNAC in the present study may be due to the sample from only the TB lymphadenitis suspected patients.

Conclusion

The fine needle aspiration cytology (FNAC) is a more sensitive test for the diagnosis of TB lymphadenitis than ZN. The FNAC showed a moderate agreement with the GeneXpert assay. This study recommends the FNA GeneXpert MTB/RIF test in preference to FNAC for the diagnosis of TB lymphadenitis to avoid a missed diagnosis of smear-negative TB lymphadenitis.

Data availability

The raw data sets used and/analyzed in the current study are available from the corresponding author upon reasonable request.

Abbreviations

TB:

Tuberculosis

TBL:

TB lymphadenitis

EPTB:

Extrapulmonary TB

FNAC:

Fine Needle Aspirate Cytology

WHO:

World Health Organization

SOP:

Standard operating procedure

AHMC:

Adama Hospital Medical College

PPV:

Positive Predictive Value

NPV:

Negative Predictive Value

References

  1. Brizi M, Celi G, Scaldazza A, Barbaro B. Diagnostic imaging of abdominal tuberculosis: gastrointestinal tract, peritoneum, lymph nodes. Rays. 1998;23(1):115–25.

    CAS  PubMed  Google Scholar 

  2. Holden IK, Lillebaek T, Andersen PH, Bjerrum S, Wejse C, Johansen IS, editors. Extrapulmonary tuberculosis in Denmark from 2009 to 2014; characteristics and predictors for treatment outcome. Open Forum Infectious Diseases; 2019: Oxford University Press US.

  3. Mathiasen VD, Eiset AH, Andersen PH, Wejse C, Lillebaek T. Epidemiology of tuberculous lymphadenitis in Denmark: a nationwide register-based study. PLoS ONE. 2019;14(8):e0221232.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Cohen A, Mathiasen VD, Schön T, Wejse C. The global prevalence of latent tuberculosis: a systematic review and meta-analysis. Eur Respir J. 2019;54(3).

  5. Bagcchi S. WHO’s global tuberculosis report 2022. Lancet Microbe. 2023;4(1):e20.

    Article  PubMed  Google Scholar 

  6. Sharma SK, Mohan A, Kohli M. Extrapulmonary Tuberculosis. Expert Rev Respir Med. 2021;15(7):931–48.

    Article  CAS  PubMed  Google Scholar 

  7. Rathi V, Ish P. Tubercular lymphadenopathy for postgraduates–A minireview. J Adv Lung Health. 2023;3(2):82–6.

    Article  Google Scholar 

  8. Gopalaswamy R, Dusthackeer VA, Kannayan S, Subbian S. Extrapulmonary tuberculosis—an update on the diagnosis, treatment and drug resistance. J Respiration. 2021;1(2):141–64.

    Article  Google Scholar 

  9. Zumla A, George A, Sharma V, Herbert RHN, Oxley A, Oliver M. The WHO 2014 global tuberculosis report—further to go. Lancet Global Health. 2015;3(1):e10–2.

    Article  PubMed  Google Scholar 

  10. Katsnelson A. Beyond the breath: exploring sex differences in tuberculosis outside the lungs. Nat Med. 2017;23(4):398–402.

    Article  CAS  PubMed  Google Scholar 

  11. Ganchua SKC, White AG, Klein EC, Flynn JL. Lymph nodes—the neglected battlefield in tuberculosis. PLoS Pathog. 2020;16(8):e1008632.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Organization WH. Xpert MTB/RIF implementation manual: technical and operational ‘how-to’; practical considerations. World Health Organ, 2014 9241506709.

  13. Organization WH. WHO meeting report of a technical expert consultation: non-inferiority analysis of Xpert MTB/RIF Ultra compared to Xpert MTB/RIF. World Health Organization; 2017.

  14. Tadesse M. Improving the diagnosis of tuberculosis in hard to diagnose populations: clinical evaluation of GeneXpert MTB/RIF and alternative approaches in Ethiopia. University of Antwerp; 2018.

  15. Mistry Y, Ninama GL, Mistry K, Rajat R, Parmar R, Godhani A. Efficacy of fine needle aspiration cytology, Ziehl-Neelsen stain and culture (Bactec) in diagnosis of tuberculosis lymphadenitis. Natl J Med Res. 2012;2(01):77–80.

    Google Scholar 

  16. Kumbi H, Reda DY, Solomon M, Teklehaimanot A, Ormago MD, Ali MM. Magnitude of tuberculosis lymphadenitis, risk factors, and rifampicin resistance at Adama City, Ethiopia: a cross-sectional study. Sci Rep. 2023;13(1):15955.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Fantahun M, Kebede A, Yenew B, Gemechu T, Mamuye Y, Tadesse M, et al. Diagnostic accuracy of Xpert MTB/RIF assay and non-molecular methods for the diagnosis of tuberculosis lymphadenitis. PLoS ONE. 2019;14(9):e0222402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. McHugh ML. Interrater reliability: the kappa statistic. Biochemia Med. 2012;22(3):276–82.

    Article  Google Scholar 

  19. Lavanya G, Sujatha C, Faheem K, Anuradha B. Comparison of GeneXpert with ZN staining in FNA samples of suspected extrapulmonary tuberculosis. IOSR J Dent Med Sci (IOSR-JDMS). 2019;18:25–30.

    Google Scholar 

  20. Singh K, Tandon S, Nagdeote S, Sharma K, Kumar A. Role of CB-NAAT in diagnosing mycobacterial tuberculosis and rifampicin resistance in tubercular peripheral lymphadenopathy. Int J Med Res Rev. 2017;5(03):242–6.

    Article  Google Scholar 

  21. Nur T, Akther S, Kamal M, Shomik M, Mondal D, Raza M. Diagnosis of Tuberculous Lymphadenitis using fine needle aspiration cytology: a comparison between Cytomorphology and GeneXpert Mycobacterium Tuberculosis resistant to rifampicin (MTB/RIF) test. Clin Infect Immun. 2019;1(01):4.

    Google Scholar 

  22. Derese Y, Hailu E, Assefa T, Bekele Y, Mihret A, Aseffa A, et al. Comparison of PCR with standard culture of fine needle aspiration samples in the diagnosis of tuberculosis lymphadenitis. J Infect Developing Ctries. 2012;6(01):53–7.

    Article  CAS  Google Scholar 

  23. Ligthelm LJ, Nicol MP, Hoek KG, Jacobson R, Van Helden PD, Marais BJ, et al. Xpert MTB/RIF for rapid diagnosis of tuberculous lymphadenitis from fine-needle-aspiration biopsy specimens. J Clin Microbiol. 2011;49(11):3967–70.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Hafez NH, Tahoun NS. Reliability of fine needle aspiration cytology (FNAC) as a diagnostic tool in cases of cervical lymphadenopathy. J Egypt Natl Cancer Inst. 2011;23(3):105–14.

    Article  Google Scholar 

  25. Mayura S, Gaddam P, Cherian S, Chaturvedi U, Naidu R. Tuberculous lymphadenitis: analysis of Cytomorphological features with utility of Genexpert MTB/RIF and other Microbiological Test as an Adjunct to Cytology. SAS J Med. 2023;8:854–9.

    Article  Google Scholar 

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Acknowledgements

We would like to thank all staffs of AHMC who was working in the laboratory for their support during laboratory examination. We also thank all the study participants for their willingness to participate in the study.

Funding

There was no specific funding received for this study.

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Authors and Affiliations

  1. Department of Laboratory Science, Adama Hospital Medical College, Po Box 84, Adama, Ethiopia

    Hawi Kumbi

  2. School of Medical Laboratory Science, College of Medicine and Health Sciences, Hawassa University, Po Box 1560, Hawassa, Ethiopia

    Musa Mohammed Ali

  3. Department of Medical Laboratory Science, College of Health Sciences, Oda Bultum University, Po Box 226, Chiro, Ethiopia

    Alegntaw Abate

Authors
  1. Hawi Kumbi
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Contributions

H.K. designed the experiment, laboratory work, data analysis and manuscript preparation, M.M.A. involved in laboratory method selection, data analysis and manuscript preparation and A.A. involved in data analysis and manuscript preparation. All authors have read and approved the manuscript.

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Correspondence to Hawi Kumbi.

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Kumbi, H., Ali, M.M. & Abate, A. Performance of fine needle aspiration cytology and Ziehl-Neelsen staining technique in the diagnosis of tuberculosis lymphadenitis. BMC Infect Dis 24, 633 (2024). https://doi.org/10.1186/s12879-024-09554-z

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BMC Infectious Diseases

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