International Journal of Biomedical Research
ISSN: 0976-9633 (Online) Journal DOI:10.7439/ijbr CODEN:IJBRFA
Research Article A comparative study of ziehl-nelson staining and auramine staining in sputum sample for the diagnosis of pulmonary tuberculosis Upasana Bhumbla*1 and Gyaneshwari2 Post Graduate Student, Department of Microbiology, Shadan Institute of Medical Sciences, Hyderabad, India Professor and Head, Department of Microbiology, Shadan Institute of Medical Sciences, Hyderabad, India.
*Correspondence Info:
Dr. Upasana Bhumbla, Post Graduate Student, Department of Microbiology, Shadan Institute of Medical Sciences, Hyderabad, India E-mail:
[email protected]
Abstract
Tuberculosis is an ancient disease, TB DNA detected by molecular analysis of a mummy dated 1550-1080BC.In developing countries where there are large number of cases and financial constraints, rapid and low cost diagnostic techniques are being introduced for the demonstration of acid fast bacilli in the sputum smears .Fluorescent microscopy is being preferred over conventional light microscopy as it is more efficient and can help in diagnosing TB at the earliest. 300 sputum samples were collected from 150 patients who were subjected to both ZN stain and Auramine O stain. Out of which 32 (10.66%) were ZN positive and 55(18.33%) were Auramine O positive. Hence it proved the efficacy and sensitivity of fluorescent microscopy over conventional light microscopy. Keywords: Ziehl Nelson, Auramine O, Fluorescent microscope
1. Introduction
Tuberculosis is declared a global emergency by WHO in 1993 and with the onset of HIV, it has taken an epidemic form. Conventional and molecular methods are available for its diagnosis. Emergence of multidrug resistant strains poses further difficulties in treatment. Hence rapid and more accurate methods are required for early diagnosis of pulmonary tuberculosis. Tuberculosis is worldwide public health problem inspite of the fact that the causative organism Mycobacterium tuberculosis wa s discovered 100 years ago. 1 Globally, it is estimated that one- third of population is asymptomatically infected with tuberculosis.2 Mostly the new cases and death occurs in the developing countries where infection is mainly acquired in the childhood. In countries with high burden of tuberculosis i t is estimated that the annual risk is 0.5-2.0 percent.3India alone accounts for one-fourth of the burden globally and the incidence and prevalence rate is seen as 185 and 256 cases per 100,000 populations respectively4.The WHO has launched a global plan for “STOP TB STRATEGY” (2006-2015), with the objective of reducing incidence of tuberculosis. This year, 2014 CDC selected the theme "Find TB. Treat TB. Working together to eliminate TB." The theme highlights that despite of decreased number of cases; TB is still a life-threatening problem. Anyone can get infected with TB, and the latest efforts for screening and treating the latent disease are not sufficient.9Sputum smear microscopy is considered as the utmost sensitive and diagnostic test available even at areas where cases are in large number and financial constraints are there. 5Fluorescent microscopy enhances the improvement of diagnosis in samples, which has a low density of these acid fast bacilli which is at times missed with ZN staining. 6 Auramine O stain is advantageous over ZN as it is simpler, cost effective and can be visualised even at low magnifications than ZN( 40x/100x).10Auramine O stain enters the bacterial cell wall and the bacilli appears bright, glowing yellow against dark background under UV light, whereas on ZN stain bacilli appear as pink coloured rods. Thus, fluorescent microscopy is more sensitive and takes 75% lesser time over ZN staining and it benefits the overburdened laboratories or places where resources are less .11 1.1 Aims and Objectives To study the sensitivity and benefits of fluorescent microscopy over conventional ZN staining.
2. Materials and Methods
300 sputum samples irrespective of all age groups were collected from 150 patients suspected with pulmonary tuberculosis including the immunocompromised patients who came to the outpatient department of Shadan Hospital. A comparative study was done from January – March, 2014 in Shadan Hospital. Two sputum samples were collected; one spot and the second one early morning fasting sample. It was collected in a sterile, c lean; wide mouth container .Each sample was processed. In Ziehl–Nelsen staining procedure; sputum slide is heat fixed and carbol fuschin is poured over the entire slide, which is kept for 5 minutes with gentle heating .It is rinsed properly and decolourised with 25% sulphuric acid, kept for 2-3 minutes.13 Slide is rinsed again and counterstained with 0.1% methylene blue for further 30 seconds. Lastly slide is washed and dried up and seen under 100x magnification of light microscope.11In Auramine O staining, flood the slide with Aura mine –phenol and keep it for 7-10 minutes. Wash well and decolorize for 2 minutes with acid alcohol, two times. It is rinsed and counterstained with 0.1% potassium permanganate with 30 seconds. Wash it well and o bserve under 40 x magnifications of fluorescent microscope.14 The results were graded as per the International Union against Tuberculosis guidelines 12.
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Upasana Bhumbla and Gyaneshwari Union /WHO scale x 1000 field =HPF Negative Scanty 1+ 2+ 3+
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Table 1: Grading chart (WHO, IUATLD, 2007) for fluorescent microscopy Bright field x 1000 magnification ; Fluorescence (x 200-250 magnification ; Fluorescence (x 400 magnification ; 1 length =2 cm=100 HPF 1 length =30 field = 300 HPF 1 length = 40 fields= 200 HPF Zero AFB / 100HPF Zero AFB / 1 length Zero AFB / 1 length 1-9 AFB / 100HPF 1-29 AFB / 1 length 1-19 AFB / 1 length 10-99 AFB / 100HPF 30-299 AFB / 1 length 20-199 AFB / 1 length 1-10 AFB / 1 HPF 10-100 AFB / 1 field on average 5-50 AFB / 1 field on average (on average 50 HPF) >10 AFB / 1 HPF >100 AFB / 1 field on average. > 50 AFB / 1 field on average (on average 20 HPF)
Fig 1. Auramine O stained sputum smear under fluorescent microscope. Bacilli appear as bright yellow organism in a dark backg round.
Fig 2. Ziehl-Nelson stained sputum smear unde r light microscope. Bacilli appear as pink coloured rod shape organism
3. Results
Out of 300 sputum samples of 150 patients, 32(10.66%) sputum samples were positive for Ziehl-Nelson and 55(18.33%) sputum samples were positive for Auramine O. Table 2: Comparison of ZN and Auramine O Staining Reports: Staining method No .of Positive smears No. of negative smears Ziehl Nelson stain 32 268 Auramine O 55 245 Figure 3: Comparison of ZN and Auramine O Staining Reports 350 300
32
55
250 200
Series 2
150
Series 1
100 50
0 Ziehl Nelson Stain
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Table 3: Comparison of led fluorescent microscopy (Auramine staining) with Ziehl nelson staining Types of staining Fluorescent microscopy Fluorescent microscopy Total methods (Auramine staining) smear positive (Auramine staining ) smear negative ZN smear positive 32 Nil 32 ZN smear negative 23 245 268 Total 55 245 300 Figure 4: Comparison of fluorescent microscopy over Ziehl nelson stain
23(7.66 32 (10.66%)
Zn and FM -ve Zn and FM +ve Zn -ve and FM +ve Zn +ve and Fm -ve
245
4. Discussion
In a study done by Ben et al in 2008, showed that out of 221 sputum samples, 33(14.9%) samples were positive with Auramine staining and 24(10.85%) samples were positive with Ziehl-Nelson staining. It demonstrated superior diagnostic results by fluorescent microscopy when compared with conventional light microscopy. Laifangbam et al in 2009 revealed a study on 102 suspected patients, where in 44.1% patients were positive with Ziehl Nelson staining and 71.6% patients were positive for Auramine O staining. It was found by this study that Auramine O staining was superior to Ziehl Nelson staining. Saroj et al in 2010 concluded in her work that the sensitivity of Auramine stain over Ziehl Nelson. It stated that out of 634 sputum samples collected, 66 (10.41%) and 105(16.56%) sputum samples were found positive for AFB by ZN and AO respectively. Where in the present study culminates that the Auramine O staining is superior to that of the conventionally used ZN stain. It states that out of 300 sputum samples; 32 (10.66%) samples were ZN positive and 55(18.33%) samples were Auramine O positive. The diagnostic accuracy of fluorescent microscope was found much more superior and much more sensitive than the conventional light microscopy. It is therefore recommended that fluorescent microscope be phased an alternative to conventional ZN light microscopy in both high and low level laboratories.7
5. Conclusion
Since the burden of tuberculosis is still a major threat for the human race. So the newer methods have been advocated where t here is a need for examination of large number of sputum smears. Hence fluorescent microscopy plays a pivotal role in today’s world for the examination of sputum smears as it is more sensitive over the conventional ZN staining. For fast and more accurate diagnosis of the disease fluorescent micro scopy appears to be more efficient and cost effective.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Park K., textbook of preventive and social medicine, 22nd edition, 2013 WHO (2011), Policy framework for implementing new tuberculosis diagnostics, March 2010. WHO (2004), Weekly Epidemiological Record, 23 Jan 2004, No.4 WHO (2012), Tuberculosis Global Facts 2011-2012. Ben J. Marias, Wendy Brittle, Katrien Painczyk, Anneke C. Hesseling, Nulda Beyers, Elizabeth Wasserman, Dick van Soolingen and Rob M Warren; Use of Light –emitting diode fluorescence microscopy to detect acid fast bacilli in sputum. Clinical infectious diseases 2008; 47:203-7. Saroj Golia, Vivek Hittinahalli, Nirmala A R, Sangeeta K.T, Asha S Kamath B. A comparative Ziehl- Nelson staining in the diagnosis of pulmonary tuberculosis. Laifangbam S, Singh HL, Singh NB, Devi KM, Singh NT; A comparative study of fluorescent microscopy with Ziehl Nelson staining and culture for the diagnosis of pulmonary tuberculosis; Kathmandu Univ Med J (KUMJ), 2009 Jul-Sep;7 (27):226-30. Khatun Z, Hossain M S, Roy CK, Sulatana T, Rahman MQ, Samad MBA Azad, Ahmad ANN: Light emitting diode (LED) Fluorescent microscopy: a Milestone in the detection of paucibacillary Mycobacterium in case of pulmonary tuberculosis.; Bangladesh Medical journal 2011: 40 (1):22-26. Centre for disease control and prevention, division of tuberculosis elimination, March 21, 2014. http://www.cdc.gov/features/worldtbday. WHOGlobaltuhttp://whqlibdoc.who.int/publications/2009/9789241563802_eng_doc.pdfberculosis control, surveillance, planning, financing, WHO report 2009. Chandra T Jaya, Selvaraj R, Sharma YV. Comparison of Variants of Carbol Fuchsin & Phenol in Ziehl Neelsen Staining to Detect AFB. J Mycobac Dis 2013; 3: 131. doi:10.4172/2161-1068.1000131. International union against tuberculosis guidelines for fluorescent microscopy by WHOM, 2007. http://www.theunion.org/ http://www.tbcindia.nic.in/pdfs/Module%20for%20Laboratory%20Technician.pdf. http://tbcindia.nic.in/pdfs/Flourescence_Microscopy%20Manual.pdf
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