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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 10  |  Issue : 3  |  Page : 136-139

Comparison of Gabbet's staining, Ziehl–Neelsen staining with fluorescent microscopy for diagnosis of pulmonary tuberculosis: A cross-sectional study


Department of Microbiology, KLE Academy of Higher Education and Research, Jawaharlal Nehru Medical College, Belagavi, Karnataka, India

Date of Submission30-Sep-2019
Date of Decision01-Mar-2021
Date of Acceptance01-Mar-2021
Date of Web Publication13-Sep-2021

Correspondence Address:
Jyoti M Nagmoti
Professor, Department of Microbiology, KLE Academy of Higher Education and Research, Jawaharlal Nehru Medical College, Belagavi, Karnataka 590010
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JCSR.JCSR_106_19

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  Abstract 


Background: Sputum staining for acid-fast bacilli and microscopic examination methods are more sensitive and specific, less time consuming, low cost than other methods for the diagnosis of pulmonary tuberculosis (TB).
Methods: In this cross-sectional study conducted over 1-year (n = 109), the sensitivity, specificity and cost-effectiveness of Gabbet's staining, Ziehl–Neelsen (Z-N) staining for the diagnosis of pulmonary TB were studied considering with fluorescence staining method as the gold standard.
Results: Majority of the patients belonged to the age group between 21 and 40 years (45.5%). With fluorescence staining, it was found that 25 of the 44 positive smears were of good quality, 12 were of average quality and 7 had poor quality. With Z-N staining, 9/29 of the positive smears were of good quality, 10 were of average quality and poor quality each. With Gabbet's staining, 12 of the 33 positive smears were of good quality, 14 were of average quality and 7 were of poor quality. The sensitivity of the Gabbet's staining (75%) was found to be greater than the Z-N staining (65.9%) considering fluorescence staining as the gold standard.
Conclusions: Gabbet's staining can be used as an effective alternative to Z-N staining in low resource settings.

Keywords: Acid-fast bacilli, diagnosis, fluorescence microscopy, Gabbet's staining, pulmonary, staining, tuberculosis, Ziehl–Neelsen method


How to cite this article:
Sharma S, Nagmoti JM. Comparison of Gabbet's staining, Ziehl–Neelsen staining with fluorescent microscopy for diagnosis of pulmonary tuberculosis: A cross-sectional study. J Clin Sci Res 2021;10:136-9

How to cite this URL:
Sharma S, Nagmoti JM. Comparison of Gabbet's staining, Ziehl–Neelsen staining with fluorescent microscopy for diagnosis of pulmonary tuberculosis: A cross-sectional study. J Clin Sci Res [serial online] 2021 [cited 2022 Jan 28];10:136-9. Available from: https://www.jcsr.co.in/text.asp?2021/10/3/136/325815




  Introduction Top


Tuberculosis (TB) is a major global health problem.[1],[2],[3] Laboratory diagnosis of TB is of crucial importance for TB control. With the advent of acid-fast staining, it was possible to demonstrate acid-fast bacilli (AFB) as diagnostic evidence of TB. Even in the present era of molecular diagnosis, microscopic examination for AFB is still relevant as it is an economical diagnostic option in resource-limited settings.[3] There are various methods which can detect AFB from clinical samples. The Gabbet's staining method[4] has been considered to have certain advantages in the field setting. We aimed to study the sensitivity, specificity and cost effectiveness of Gabbet's, staining, Ziehl–Neelsen (Z-N) staining in a microbiology laboratory at a medical college teaching hospital in South India.


  Material and Methods Top


It is the cross-sectional study; permission to conduct the study was obtained from institutional Ethics Committee on Human Subjects' Research of Jawaharlal Nehru Medical College, Belagavi. The study was conducted on sputum samples from patients clinically suspected to have pulmonary TB that were received at the microbiology laboratory and Revised National TB Control Programme (RNTCP) Designated Microscopy Centre, Dr. Prabhakar Kore Charitable Hospital Belagavi Karnataka, India, from January to December 2018. Patients known to have any other chest diseases were excluded from the study.

Early morning sputum samples were collected according to the RNTCP guidelines.

The patients were given clean, sterile, leak-proof, wide mouth containers on his/her first attendance. They were instructed to collect the sample as per the following steps: The patient was asked to take a deep breath for two to three times; Then cough out deep from the chest; Open the container and spit the sputum into a universal sterile container; Close the container. If sputum was mucoid then only it is accepted, if only saliva is present it should be rejected. A good specimen should be thick, mucopurulent and sufficient quantity (5 mL). After labelling the collected specimen was transported to the laboratory as soon as possible. If there was a delay in transporting the specimens they were refrigerated to inhibit the growth of the unwanted microorganisms.

Gross examination of sputum was done specifically noting the consistency and appearance: normal/viscoelastic, colour: (yellow/green/red tinged) and odour (normal/putrid). The prepared smear was stained and observed under bright field microscopy, Z-N stain[5] fluorescence microscopy [Table 1][6] and Gabbet's staining.[4] The Gabbet's stain is used as an alternative to Z-N stain in a bright field microscope for the detection of AFB. The principle of Gabbet's staining is same as Z-N staining. By fluorescence staining technique, slides can be examined at a lower magnification that allows it to examine much larger area per unit time. The area that requires 10 min to examine by Z-N stain can be examined in 2 min by fluorescence microscopy.
Table 1: Sputum smear grading on Ziehl–Neelsen and fluorescent staining

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Gabbet's staining carbolfuchsin used as a primary stain and Gabbet's methylene blue is used as both decolouriser as well as a counter stain. Compared with Z-N stain, Gabbet's stain is less time consuming and simple to use. It does not need the heating step in staining procedure (hence called 'cold staining') and also does not require the decolourising step. It is economical and less cumbersome as well.

As per the RNTCP guidelines,[5],[6] there is a different grading system according to the number of bacilli present in the different numbers of fields in bright field and fluorescence microscopy [Table 1]. The grading of the smear was also done according to the quality of staining [Table 2].
Table 2: Quality analysis grading

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Statistical analysis

Considering fluorescence staining as the 'gold standard', the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Z-N stain and Gabbet's stain were calculated.


  Results Top


During the study, of 109 sputum samples were collected; there were 68 (62.4%) males. Majority of the patients belonged to the age group between 21 and 40 years (45.5%). Further, the positive smears were studied for qualitative measures, such as, bacterial morphology, background quality and clarity of smear and they were classified as good, average and poor. With fluorescence staining, it was found that 25 of the 44 positive smears were of good quality, 12 were of average quality and 7 had poor quality. With Z-N staining, 9 of the 29 positive smears were of good quality, 10 were of average quality and poor quality each. With Gabbet's staining, 12 of the 33 positive smears were of good quality, 14 were of average quality and 7 were of poor quality.

The sensitivity, specificity, PPV, NPV of Z-N staining and Gabbet's staining considering fluorescence staining as 'gold standard' is shown in [Table 3]. Gabbet's staining had a higher sensitivity (75% vs. 65.9%) and similar specificity compared with Z-N stain [Table 3]. Cross-tabulation of AFB grading by fluorescence, Z-N and Gabbet's staining methods is shown in [Table 4].
Table 3: Sensitivity, specificity, positive predictive value, negative predictive value of acid-fast bacilli staining as per Ziehl-Neelsen staining and Gabbet's staining positivity

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Table 4: Acid-fast bacilli grading by fluorescence staining, Ziehl–Neelsen stain and Gabbet's stain

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  Discussion Top


In the Z-N method, the basic fuchsin phenol dye is used hot. This results in melting of the unsaponifiable waxy substance on the surface of the cell wall. The Z-N staining is a cumbersome method because it requires heat application during the carbolfuchsin staining and, therefore, the need for a flame source. In the cold staining methods developed for the demonstration of AFB have been designed by eliminating heating step of Z-N technique[7]. In a cold staining method, the Gabbet's staining method, methylene blue acts as decolouriser and counter stain, it has been advocated as an alternative staining technique.

The present study aimed to compare the performance of Z-N stain, Gabbet's stain with fluorescence stain. The smears were prepared from the samples and observed by both bright field microscopy and fluorescence microscopy. Age wise distribution of samples in our study were distributed into five groups, we found majority of them belonged to age group between 21-40 years (45.5%). In another study[8] majority of AFB (48.7%) positive were observed to be in the age group between of 15- 30 years. The present study showed male preponderance among pulmonary TB patients. Similar results were reported in another study[8],[9].

Our study showed that majority of subjects were positive by fluorescence staining 44 (40.4%) followed by Gabbet's staining 33 (30.3%) and Z-N staining 29 (26. 6%). Similar reports were documented in another study[8] in which out of total 176 sputum samples, 68 (38.6%) were positive by fluorescence stain and 35 (19.9%) were positive by Z-N stain method. In a study[10] conducted in Navi Mumbai, number of smears positive for AFB by ZN method, Gabbet's method and fluorescent method were 13 (16%), 7 (9%) and 26 (33%) respectively.

Fluorescence staining method was found to be more efficient as compared to ZN staining. In another study[10] the authors also found that the fluorescence staining was more sensitive as compared to Z-N staining, which is in accordance with the observations documented in our study.

Another study[11] 200 sputum samples from suspected cases of pulmonary tuberculosis from Santhiram General Hospital, Nandyal, Andhra Pradesh, India were studied comparing Gabbet's, fluorescent staining with Z-N staining. The authors[11] reported that 18.5% were positive by Z-N method, 16.5% were positive by Gabbet's staining and 23.5% were positive by fluorescent staining method.

In the present study Gabbet's staining was found to be more sensitive than the Z-N staining (75% Vs 65.9%). In another study[12] Z-N stain and Gabbet's staining had a sensitivity 83.5% and 62.2% respectively considering fluorescence staining as the gold standard. In our study the NPV for Z-N stain was 81.25% compared with 85.5% observed for Gabbet's stain.

In a study[13] from Egypt, the authors evaluated three different staining techniques used in the detection of AFB, namely, the conventional ZN stain and two cold stains: Gabbett's cold stain and modified cold stain (MCS). Considering culture as the gold standard, the highest sensitivity (76.6%) and specificity (98.5%) were seen for Gabbett's stain. ZN stain had a sensitivity and specificity of 70 and 97.1% respectively and modified cold stain had a sensitivity and specificity of 60% and 95.7%, respectively. In a study[14] from Manipal, of the 260 samples, 16 (6.2%), 15 (5.8%), and 13 (5%) tested positive for AFB results with fluorescent staining, modified cold staining, and Gabbet's staining, respectively. The sensitivity of Gabbet's staining and modified cold staining, in comparison with that of FS, was 81.3% and 93.8%, respectively. The concordance of Gabbet's staining (0.988) and modified cold staining (0.996) with fluorescent staining was good.

The present study analyzed the quality of the stained smears and was graded as 'good' (3+), 'average' (2+) and 'poor' (1+) on the basis of appearance of the bacterial morphology, background quality and clarity of smear. We found 46 smears could be categorized into good, 36 into average and 24 into poor quality. This categorization is first of a kind for assessment of the quality of smears which helped in proper analysis and comparison between various staining techniques used in the present study. The utility of this system needs to be validated in future studies with an appropriate sample size.

Another recent prospective study (15) conducted in 266 sputum samples received at the DMC at a government tertiary care hospital were subjected to Z-N staining modified cold stain and fluorescent microscopy. The authors [15] reported that the concordance of results between Z-N and modified cold method was 97.3%. The authors reported that the modified cold method was economical and safe and can be adopted for training purposes.

Our observations suggest that Gabbet's staining can be used in low resource settings as an economic and easy to perform method for demonstrating AFB.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
World Health Organization. Global tuberculosis report 2020. Geneva: World Health Organization; 2020.  Back to cited text no. 1
    
2.
Goyal R, Kumar A. A comparison of Ziehl-Neelsen staining and fluorescent microscopy for diagnosis of pulmonary tuberculosis. IOSR J Dent Med Sci 2013; 8:05-8.  Back to cited text no. 2
    
3.
Dezemon Z, Muvunyi CM, Jacob O. Staining techniques for detection of acid-fast bacilli: what hope does fluorescein-diacetate (FDA) vitality staining technique represent for the monitoring of tuberculosis treatment in resource-limited settings. Int Res J Bacteriol 2014;1:1.  Back to cited text no. 3
    
4.
Vasanthakumari R, Jagannath K, Rajasekaran S. A cold staining for acid fast bacilli. Bull World Health Organ 1986;64:741-3.  Back to cited text no. 4
    
5.
Central TB Division, Ministry of Health and Family Welfare, Government of India. Revised National TB Control Programme. Manual for laboratory technicians. Smear microscopy for detection of acid-fast bacilli. Available atURL: http://tbcindia.gov.in/WriteReadData/l892s/6341531217Module%20for%20Laboratory%20Technician.pdf. Accessed January 2, 2021.  Back to cited text no. 5
    
6.
Central TB Division, Ministry of Health and Family Welfare, Government of India. Revised National TB Control Programme. Manual for fluorescence microscopy. Available at URL: http://tbcindia.nic.in/WriteReadData/l892s/7890638455Flourescence_Microscopy%20Manual.pdf. Accessed January 2, 2021.  Back to cited text no. 6
    
7.
Lahiri KK, Chatterjee SK. A simple cold staining method for acid fast bacilli. Med J Armed Forces India 1994;50:256-8.  Back to cited text no. 7
    
8.
Desai K, Malek S, Mehtaliya C. Comparative study of ZN staining v/s Fluorochrome stain from pulmonary and extra-pulmonary tuberculosis. Gujarat Med J 2009;64:32-4.  Back to cited text no. 8
    
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Ramakrishna R, Chundari B. A comparative study of sputum smears by Ziehl Neelsen staining versus fluorescent staining in the diagnosis of pulmonary tuberculosis in patients suffering with cough for 2 weeks or more. J Evid Based Med Healthcare 2015;2:1663-74.  Back to cited text no. 9
    
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Manta R, Prabil A, Dalal PJ. of staining techniques–Ziehl Neelsen stain, Gabbet's Stain, Fluorochrome stain for detection of mycobacterium tuberculosis in sputum. Indian J Microbiol Res 2018;5:44-6.  Back to cited text no. 10
    
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Balakrishna J, Shahapur PR, Chakradhar P, Saheb HS. Comparative study of different staining techniques – Ziehl Neelsen stain, Gabbet's stain, fluorochrome stain for detecting Mycobacterium tuberculosis in the sputum. Int J Pharm Sci Res 2013;5:89.  Back to cited text no. 11
    
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Priya S, Kuruvilla T.S. A comparative study of Ziehl Neelsen, Gabbet's and fluorescent staining methods to detect acid fast bacilli in sputum samples. GJRA. 2018;7:29-30.  Back to cited text no. 12
    
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Abdelaziz MM, Bakr WM, Hussien SM, Amine AE. Diagnosis of pulmonary tuberculosis using Ziehl-Neelsen stain or cold staining techniques? J Egypt Public Health Assoc 2016;9:39-43.  Back to cited text no. 13
    
14.
Gupta S, Shenoy VP, Bairy I, Muralidharan S. Comparison among three cold staining methods in the primary diagnosis of tuberculosis: a pilot study. J Bras Pneumol. 2010;36:612-6.  Back to cited text no. 14
    
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Madhusudhan NS, Amirthalingeswaran G. Comparison of ZN stain (RNTCP) versus fluorescent microscopy and modification of cold stain to detect acid fast bacilli from sputum sample. Int J Contemp Med Res 2016;3:968-71.  Back to cited text no. 15
    



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



 

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