Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 
Print this page Email this page Users Online: 78

 Table of Contents  
Year : 2019  |  Volume : 8  |  Issue : 4  |  Page : 238-243

Comparison of Ziehl-Neelsen's stain, fluorescent stain with CBNAAT of sputum for the diagnosis of pulmonary tuberculosis

Department of Microbiology, Kakatiya Medical College, Warangal, TS, Telangana, India

Date of Submission25-Mar-2019
Date of Decision28-Sep-2019
Date of Acceptance29-Oct-2019
Date of Web Publication16-Dec-2019

Correspondence Address:
Dr. Goteti V Padmaja
Associate Professor and HOD, Department of Microbiology, Kakatiya Medical College, Warangal, TS, Telangana
Login to access the Email id

Source of Support: None, Conflict of Interest: None


Rights and Permissions

Introduction: Pulmonary tuberculosis remains one of the most dangerous communicable diseases. More than two billion people are estimated to be infected with Mycobacterium tuberculosis. In 2017, approximately 10 million individuals became ill with tuberculosis,among them 2.79 million individuals were active cases, 1.6 million died among which 0.3 million died with coexistent HIV infection. Prompt diagnosis of active tuberculosis facilitates timely therapeutic intervention and minimize the community transmission. As tuberculosis is a global problem, for eradication of the disease, early diagnosis, timely identification and improved detection is essential. The diagnosis of tuberculosis is challenging in cases of insufficient sputum and paucity of bacilli. With this background, this study was planned to compare Ziehl-Neelsen's stain, Fluorescent stain and CBNAAT (Cartridge based nucleic acid amplification test) as per RNTCP guidelines.
Material and Methods: A prospective interventional study was carried out using 188 sputum samples of suspected pulmonary tuberculosis. All 188 samples were subjected to three methods Ziehl-Neelsen's stain, fluorescent stain and CBNAAT. They were compared for sensitivity and specificity in terms of qualitative results. The data recorded was then analyzed statistically.
Results: In our study out of 188 cases 124 were males, 64 were females. Out of 188 samples, the smear positivity rate of ZN stain was 33.5%, fluorescent stain was 40.9%, and the positivity rate of CBNAAT was 51.1%.
Conclusion: CBNAAT is more sensitive and specific in detection of pulmonary TB. An additional feature of rifampicin resistance can also be detected by CBNAAT.

Keywords: Acid fast bacilli, CBNAAT, fluorescent stain, pulmonary tuberculosis, Ziehl-Neelsen stain

How to cite this article:
Padmaja GV, Srujana K, Sadhana C. Comparison of Ziehl-Neelsen's stain, fluorescent stain with CBNAAT of sputum for the diagnosis of pulmonary tuberculosis. J NTR Univ Health Sci 2019;8:238-43

How to cite this URL:
Padmaja GV, Srujana K, Sadhana C. Comparison of Ziehl-Neelsen's stain, fluorescent stain with CBNAAT of sputum for the diagnosis of pulmonary tuberculosis. J NTR Univ Health Sci [serial online] 2019 [cited 2020 Oct 1];8:238-43. Available from: http://www.jdrntruhs.org/text.asp?2019/8/4/238/273130

  Introduction Top

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. In 2017, approximately 10 million individuals became ill with TB, among them 2.79 million individuals were active cases. 1.6 million died, among which 0.3 million died with coexistent HIV infection.[1] The diagnosis of pulmonary tuberculosis should be suspected in patients with relevant clinical manifestations (cough >2 to 3 weeks duration, lymphadenopathy, fever, night sweats, weight loss) and relevant epidemiologic factors (history of prior TB infection or disease, known or possible TB exposure, and/or past or present residence in or travel to an area where TB is endemic). Patients being evaluated for pulmonary tuberculosis who pose a public health risk for transmission should be admitted and isolated with airborne precautions. According to WHO, tubercular infections are currently spreading at the rate of one person per se cond per million people in our country.[2] There are various methods of bacteriological diagnosis of TB. Tuberculosis commonly affects lungs but can also be extra pulmonary. Hence, microscopic examination of sputum for detection of acid fast bacilli is of utmost importance. Early detection can prevent further complications. As per Revised National Tuberculosis Control Program (RNTCP) or World Health Organization (WHO), an individual with at least one sputum smear positive for AFB or culture positive for tubercle bacilli is labeled to be suffering from pulmonary tuberculosis. Most of the TB cases are diagnosed based on sputum smear microscopy. The search for rapid and efficient method has resulted in several modifications of Ziehl-Neelsen's stain and fluorescent stain. Microbiological diagnosis is essential for effective and prompt treatment of pulmonary tuberculosis. WHO also recommended a Real- time PCR test (CBNAAT) as a primary diagnostic modality.[3],[4]

  Methods Top

Quality specimens are vital for the laboratory diagnosis of TB and Sputum is the most frequent specimen collected for TB testing. Sputum is a respiratory secretion originating from deep within the lungs. Patients are instructed on the difference between sputum and saliva or nasopharyngeal secretions and the necessity for a deep, productive cough. Sputum specimens are collected under the direction of a trained healthcare professional. Specimens are collected in sterile, clear, plastic, leak-proof and wide-mouthed sterile containers. The comprehensive study was conducted at MGM hospital where a total of 188 sputum samples were collected and subjected to Ziehl-Neelsen's stain, fluorescent stain and CBNAAT.[5] 100 sputum samples taken as controls were negative by Ziehl-Neelsen's stain, fluorescent stain and CBNAAT [Table 1].
Table 1: Distribution of Pulmonary Tuberculosis Cases

Click here to view

The diagnosis of Pulmonary Tuberculosis by demonstration of acid fast bacilli in sputum smears by ZN stain is cost effective.[6] They stain with carbol fuchsin, which is a red dye. They retain the dye when treated with acid, because of the presence of mycolic acid in their cell wall. The decolorizing agent used is 20% H2 SO4.

Fluorescent stain by Auramine is another method of staining technique. In this technique, the smear is made from the specimen and stained with fluorescent stain called Auramine. The auramine stain enters the bacterial cell wall and makes them glow against dark background under UV light. Microscopic examination under low power objective will reveal mycobacterium as glowing yellow-white-like bacteria in the smear.[7]

Cartridge based nucleic acid amplification test (CBNAAT) is a recently introduced polymerase chain reaction based method for detection of TB. It is a MTB specific automated, cartridge based nucleic acid amplification assay, having fully integrated and automated amplification and detection using real time PCR, providing results within 100 minutes.[8] It has a highly specific primer and five unique molecular probes to target the rpoB gene of M. tuberculosis, which is the critical gene associated with rifampicin resistance.[9] No cross reactions have been observed with many other bacterial species tested, including a comprehensive band of mycobacteria, there by excluding non-tubercular bacteria.

Inclusion criteria

All adults of both gender suspected to be a case of pulmonary tuberculosis as per RNTCP guidelines were included.

Exclusion criteria

Samples other than sputum samples, macroscopically resembling saliva were excluded. Sputum samples retrieved with suspected pneumonia cases or from other lower respiratory tract infections were also excluded.

Sample collection method

For initial diagnosis of pulmonary tuberculosis, collect a series of three sputum specimens 8-24 hours apart, at least one of which is an early morning specimen. Optimally, specimens are collected before drug therapy is started as even a few days of treatment may inhibit growth and prevent isolation of M. tuberculosis complex (MTBC).[10]

Samples submitted for the initial diagnosis of TB should be tested by both Ziehl-Neelsen's stain, Fluorescent stain and CBNAAT. It is recommended that Nucleic acid amplification (NAA) testing be performed on at least one respiratory specimen from each patient with signs and symptoms of pulmonary TB. All sputum samples received for CBNAAT are subjected to. All sputum samples received for CBNAAT were subjected to Ziehl-Neelsen's stain and fluorescent stain and all the smears were examined microscopically.[11],[12]

Ziehl-Neelsen's stain

The fixed smears were stained with Ziehl-Neelsen's stain and visualized under an oil immersion microscope (100 × magnification). Each slide was observed for 5-10 minutes, corresponding to 300 fields examined. Samples which were positive and negative for AFB were subjected to fluorescent stain and CBNAAT [Figure 1].
Figure 1: ZN Staining positive for M. TB

Click here to view

Fluorescent stain

For the smear examination, fixed preparations were stained with Auramine-o stain and examined under fluorescent microscope [Figure 2].
Figure 2: Fluorescent staining positive for M.TB

Click here to view

  Cbnaat Top

For CBNAAT, sputum sample was poured into a single use disposable cartridge that is placed into the Xpert Dx module with the results produced in less than 2 hours. Each PCR run comprises of an internal control for sample processing [DNA extraction] and PCR validity [presence of inhibitors] with positive and negative control tested every day. The system automatically interprets all results from measured fluorescent signals with embedded calculation algorithms, into the following categories positive, negative or invalid if PCR inhibitors are detected with amplification failure. The strain was categorized as susceptible or resistant to rifampicin [Figure 3], [Figure 4], [Figure 5].
Figure 3: CBNAAT POSITIVE report for M.TB

Click here to view
Figure 4: CBNAAT NEGATIVE report for M.TB

Click here to view
Figure 5: CBNAAT: Positive report of M. Tuberculosis

Click here to view

  Results Top

In our study, a total of 188 samples were collected and subjected to Ziehl-Neelsen's stain, fluorescent stain and CBNAAT. The smear positivity rate of Ziehl-Neelsen's stain was 33.5% and for fluorescent stain was 40.9%, while the detection of MTB in CBNAAT was 51.1% [Table 2].
Table 2: Comparison of results of ZN Staining, Fluorescent Staining, CBNAAT

Click here to view

  Discussion Top

India has a long history of research and demonstration projects on TB. The detection of acid fast bacilli was often considered as the evidence of the infected stage. Thus, the laboratory plays a critical role in the diagnosis of TB. In developing countries, microscopy of the specimen is by far the fastest, cheapest, and most reliable method for the detection of AFB. However, fluorescent stain has been added in Revised National Tuberculosis Control Program (RNTCP) because of more sensitive and rapid results and can be used in field areas. In the present study, the results showed that from sputum specimen of 188, 63 patients were smear positive with ZN stain and 77 positive with fluorescent stain. These results indicate that Auramine staining of sputum smears is a more sensitive method of sputum microscopy for demonstration of AFB in sputum specimen, compared to Ziehl-Neelsen's stain. The use of fluorescent stain greatly improves the diagnostic value of sputum smear, especially in patients with low density of bacilli that are likely to be missed on Ziehl-Neelsen's stain. This method is economical in both time and cost and recommended for laboratories handling large number of sputum specimens. Fluorescent stain is superior to that of Ziehl-Neelsen's stain in the presence of a low bacterial load as seen in smears with diagnostic cytomorphological featured tuberculosis, in problem areas like AIE (acute inflammatory exudates) alone or with occasional granuloma, AFB positivity by Ziehl-Neelsen's stain is nearly as good as the fluorescent method because bacterial load is high. Using fluorescent staining, the tubercle bacilli when examined under ultraviolet illumination, the bacilli appeared as a bright rod against a dark background. Since there was a contrast, the bacilli were readily seen, and therefore in very less time, a large area could be examined. Images were then captured with the digital camera and enhance through imaging processing techniques. While in, Ziehl-Neelsen's stain acid fast bacilli appear as pink rods in blue background. The potential benefits of automated screening for tubercle bacilli are: rapid, acute, inexpensive; the ability to screen large number of people; increased resources to monitor patients; and reduction in health risk to staff.

The fact that fluorescent stain has difficulty in detecting paucibacillary can be attributed to the fact that only a loop-full of sputum sample was taken to make the smear slides. While 2 ml sputum sample for CBNAAT was used for the process [Table 3]. In a study by Navinchandra, he found out that the sodium hypochlorite concentration technique leads to 44.11% increase in detection of new cases as compare to routine RNTCP method.[13]
Table 3: Sensitivity and Specificity of Staining and CBNAAT

Click here to view

  Conclusion Top

CBNAAT is more sensitive and specific in detection of pulmonary TB. An additional feature of rifampicin resistance can also be detected by CBNAAT. Routine investigation with CBNAAT will detect early pulmonary TB as it can detect paucibacillary cases. As there is evolution of MDR bacilli and coexistence with HIV/AIDS, early detection of Pulmonary TB cases is a dire necessity for the control of global pandemic. WHO also recommended a Real-time PCR test (CBNAAT) as a primary diagnostic modality. Therefore, CBNAAT should be used for early diagnosis not to miss the paucibacillary cases as it increases rapid case detection by 10-20% and plays an important role in effective patient management and to control the subclinical spread of the disease in the community [Table 4] and [Table 5].[14],[15],[16],[17] But for maximum recovery of mycobacteria, rapid culture methods like MGIT can also be incorporated along with CBNAAT.
Table 4: Comparison of Different Studies With Present Study

Click here to view
Table 5: Advantages and Disadvantages of Znstain, Flourescentstain and CBNAAT

Click here to view


We acknowledge the support extended by superintendent and technical staff of TB and Chest diseases hospital, Naim Nagar, Warangal.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

World Health Organization (WHO). Global tuberculosis report 2018.  Back to cited text no. 1
World Health Organisation. Global TB Report 2015. Vol. 20. France: World Health Organisation; 2015. p. 14-5. Available from: https://apps.who.int/iris/bitstream/handle/10665/191102/9789241565059_eng.pdf;jsessionid. [Last accessed on 2019 Mar 01].  Back to cited text no. 2
Brisson-Noël A, Gicquel B, Lecossier D, Lévy-Frébault V, Nassif X, Hance AJ. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet 1989;2:1069-71.  Back to cited text no. 3
Centers for Disease Control and Prevention (CDC). Update: Nucleic acid amplification tests for tuberculosis. MMWR Morb Mortal Wkly Rep 2000;49:593-4.  Back to cited text no. 4
Gupta A, Sharma SK, Pande JN. Diagnostic methods for tuberculosis, the India. J Chest Dis Allied Serv 1993;35:63-84.  Back to cited text no. 5
Munir MK, Rehman S, Aasim M, Iqbal R, Saeed S. Comparison of Ziehl Neelson microscopy with GeneXpert for detection of Mycobacterium tuberculosis. IOSR J Dent Med Sci 2015;14:56-60.  Back to cited text no. 6
World Health Organization. Fluorescent light-emitting diode (LED) microscopy for diagnosis of tuberculosis: Policy statement. Geneva, Switzerland: WHO; 2011. [cited 2017 Nov 30]. Available from: http://www.who.int/tb/dots/laboratory/who_policy_led_microscopy_july10.pdf.  Back to cited text no. 7
Lawn SD, Nicol MP. Xpert® MTB/RIF assay: Development, evaluation and implementation of a new rapid molecular diagnostic for tuberculosis and rifampicin resistance. Future Microbiol 2011;6:1067-82.  Back to cited text no. 8
Helb D, Jones M, Story E, Boehme C, Wallace E, Ho K, et al. Rapid detection of mycobacterium tuberculosis and rifampicin resistance by use of on demand, near-patient technology. J Clin Microbiol 2010;48:229-37.  Back to cited text no. 9
Kasper DL, Fauci AS, Hauser S, Longo D, Jameson JL, Loscalzo J. Harrison's Principles of Internal Medicine 19/E (Vol. 1 and Vol. 2). McGraw-Hill Education; 2015.  Back to cited text no. 10
Steingart KR, Henry M, Ng V, Hopewell PC, Ramsay A, Cunningham J, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis a systematic review. Lancet Infect Dis 2006;6:570-81.  Back to cited text no. 11
Laifangbam S, Singh HL, Singh NB. A comparative study of fluorescent microscopy, Ziehl-Neelsen staining and culture for the diagnosis of pulmonary tuberculosis. KUMJ 2009;7:226-30.  Back to cited text no. 12
Navinchandra MK, Kalpana PD, Vilas RT. Increased sensitivity of sputum microscopy with sodium hypochlorite concentration technique a practical experience at RNTCP centre. Lung India 2011;28:17-20.  Back to cited text no. 13
Kumar P, Goswami P, Kumar M, Kumari R, Ali SK. Effectiveness of Xpert MTB/RIF on smear negative samples tested by LED Fluorescence microscopy for rapid diagnostics of TB cases in Bihar. J Sci Eng Res 2017;4:78-80.  Back to cited text no. 14
Sutay N, Jha S, Chaudhary D. A comperative study between Genexpert and Smear Diagnosis of Tuberculosis in Paediatric patients. Int J Sci Res 2017;6: 1616-9.  Back to cited text no. 15
Taddese BD, Desalegn DM, Misganaw AS, Kitila KT, Balcha HM, et al. Comparison of GeneXpert against Light-Emitting Diode Fluorescent Microscopy for the Diagnosis of Pulmonary Tuberculosis in Addis Ababa, Ethiopia. J Microb Biochem Technol 2018;10:12-5.   Back to cited text no. 16
Salinita N, Supriya L, Usharani M, Singh HL. Detection of pulmonary tuberculosis using cartridge based nucleic acid amplification test (CBNAAT) and fluorescent microscopy. IJHRMLP 2018;4:78-81.  Back to cited text no. 17


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Article Figures
Article Tables

 Article Access Statistics
    PDF Downloaded240    
    Comments [Add]    

Recommend this journal