|Year : 2014 | Volume
| Issue : 2 | Page : 153-156
Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry
TJ Chandra1, RS Raj2, YV Sharma3
1 Department of Microbiology, GSL Medical College, Rajahmundry, Chennai, India
2 Centre for Lab Animal Research (CLARE), Sathyabama University, Chennai, India
3 Department of Pathology, GSL Medical College, Rajahmundry, Andhra Pradesh, India
|Date of Submission||09-Mar-2013|
|Date of Acceptance||04-May-2013|
|Date of Web Publication||2-Apr-2014|
T J Chandra
Department of Microbiology, GSL Medical College, Rajahmundry, Chennai
Source of Support: None, Conflict of Interest: None
Background: Sputum smear microscopy is the main-stay in the diagnosis of pulmonary tuberculosis in many developing countries. To overcome the drop outs, same day diagnosis is ideal. Materials and Methods: In the current study, two spot sputum samples (SS 2 approach) are collected within a gap of one hour (same day sputum smear microscopy) in addition to the standard spot morning (SM) approach. The smears were stained with standard Ziehl Neelsen (ZN) and modified ZN staining techniques. Results: Out of 1537 patients, sputum smear positivity (SSP) was 9.43% (146 patients) in SM approach with standard ZN staining. Smear positivity was increased to 9.8% (151 patients) with modified ZN staining. For SS 2 approach, SSP was 9.37% (144 patients) and 9.8% (151 patients) with standard and modified ZN staining procedures, respectively. Conclusions: Diagnosis of lung tuberculosis is possible with two spot sputum samples with modified ZN staining.
Keywords: Modified ZN staining, sputum smear microscopy, standard approach, same day (SS 2 ) approach, Ziehl Neelsen staining
|How to cite this article:|
Chandra T J, Raj R S, Sharma Y V. Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry. Indian J Med Microbiol 2014;32:153-6
|How to cite this URL:|
Chandra T J, Raj R S, Sharma Y V. Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry. Indian J Med Microbiol [serial online] 2014 [cited 2020 Jan 25];32:153-6. Available from: http://www.ijmm.org/text.asp?2014/32/2/153/129801
| ~ Introduction|| |
Tuberculosis (TB) is a world pandemic, a bacterial disease caused by Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. africanum). Various diagnostic methods are available for the diagnosis of lung TB: identifying bacilli in sputum smears (Microscopy), culture on LJ medium, BACTEC, gene expert, etc., But in most of the countries pulmonary TB is diagnosed by identifying tubercle bacilli in sputum smears by Ziehl Neelsen (ZN) staining. This is a rapid, economical method. In spite of its few disadvantages such as inability to identify drug resistance, limited utility in the diagnosis of TB in human immunodeficiency virus (HIV) patients, World Health Organization (WHO) and Revised National Tuberculosis Control Programme (RNTCP) insist sputum smear microscopy (ssm).
Under most of the national TB control programmes (NTPs), lung TB is diagnosed by screening three sputum samples (spot-morning-spot (SMS) approach). This SMS approach would require 2 days time period. In SMS approach, most TB cases were diagnosed by the first two samples of sputum.  So currently lung TB diagnosis is by observing two samples of sputum (spot-morning (SM[TAG:2][/TAG:2]
scheme). This SM approach would also require 2 days time period as patient has to visit the microscopy/TB diagnostic centre for 2 days. The advantage with SM approach is reduction of the work load on laboratory technicians  as also minimising expenditure, so NTP's money can be saved to some extent, that is, on sample containers, staining reagents, glass slides, etc., Due to reduction of work load in SM approach (two sputum samples instead of three), quality of ssm may be improved. Like SMS, in SM approach also patient has to spend a significant amount of money for travelling to reach the microscopy centre, food, etc., In addition, SM approach also leads to loss of patient working days.
The major obstacle with ssm is patient dropouts. This dropout rate is usually more in field conditions. With defaults diagnosis of TB may not be done in correct time, automatically reflecting on access to treatment.  It was proved that each individual with active TB can spread the disease to 10-12 members per annum.  Thus, this global killer profoundly effects the financial statuses and foreign relations of the countries by causing the WHO and NTPs to spend significant amount.
The only alternative to overcome the dropouts is early or if possible same day diagnosis of TB. Several new, rapid TB diagnostic tests are available, which can give test results in 2-3 hours with high sensitivity and specificity, for example Line Probe Assay (LPA), Loop Mediated Isothermal Amplification (LAMP), Gene X pert. But, resource-limited and high TB-burden countries like India, rapid TB diagnostic tests are not possible due to their high cost. Hence in developing countries like India, TB diagnosis on same day by ssm is a significant alternative. The sputum smear positivity (SSP) of SM approach was 0.29% more when compared with SS 2 (same day ssm) approach in our previous study.  In SS 2 approach, two spot sputum samples are collected within one hour and these sputum smears are stained by ZN staining as per the RNTCP guidelines.  Statistically there was no significant difference (P > 0.05) between the schemes concerned (smear positivity was 9.72% and 9.43%, respectively, for SM and SS 2 approaches). But, ethically we should not miss any positive cases. By keeping this fact in mind we searched the literature for different modifications for better results of ZN staining technique.
Selvakumar et al., showed that ZN staining by using 0.1% basic fuchsin (BF) detected only 75% of the smears found positive by ZN staining using 1% BF.  Whereas in another study by Selvakumar et al.,  it was concluded that use of 0.3% of BF may result in 20% smear-positive patients being missed. Later Pawan et al.,  explained that reduced smear-positive results were due to lowered concentration of phenol to ~ 1.7%. But in the standard ZN  staining the phenol concentration is 5%.
As per the studies, , it was very clear that in ZN staining, concentration of phenol should be 5% and BF should be 1%. Otherwise the smear positivity will be reduced automatically. RNTCP,  Government of India, recommends usage of 1% BF in ZN staining, 5 min exposure time, while WHO  and the International Union Against Tuberculosis and Lung Disease (IUATLD)  guidelines recommend only 0.3% BF. Van deun et al.,  showed that ZN staining with 1% BF for 15 min might be superior to 0.3% BF for 5 min. Hence in the present study prolonged primary staining step (15 min) is used for the sputum samples of SM and SS 2 approaches to identify tubercle bacilli. The results of modified ZN staining were compared with standard ZN method.
| ~ Materials and Methods|| |
The study was conducted in the department of Microbiology, GSL Medical College, Rajahmundry from January 2011 to October 2012, over a period of 22 months. Study was approved by Institutional Research and Ethics Committee. An informed written consent in the presence of witness was taken from all the volunteers who participated in the study. Individuals aged 14 years or above were included in the study.
All the individuals were explained about the importance of submission of sputum sample. The visual difference between sputum and saliva was demonstrated. They were explained how to produce a good quality sputum sample in the local language and it was demonstrated practically. Finally they were explained to provide 5 ml of sputum sample. All the individuals were informed to provide three sputum samples (SS 2 M scheme), that is spot sample at the time of first visit to the hospital (S), S 2 is second spot collected one hour after S sample. M sample was collected after getting up from bed early in the morning. After collecting the two spot samples, patients were provided with pre-labelled sample containers to collect M samples at home.
Immediately after collection, two smears were prepared with each sample on new glass slide and one slide was stained with standard ZN technique as per RNTCP guidelines.  Second smear was stained by modified ZN method. After ZN staining slides were covered with wrap around stickers, so that the microscopist was not aware of smear staining technique, thus avoiding bias.
Preparation of staining reagents was done as per the RNTCP guidelines. 
A new unscratched slide was selected for smear preparation. Smear was prepared with sterile loop. A good smear is spread evenly, over a size of 2 × 3 cm and is neither too thick nor too thin. This was allowed to air dry for 15-30 min and fixed by passing it over a blue flame 3-4 times. 
Smears, flooded with filtered 1% carbol fuchsin (CF) were heated until it was steaming and left to steam for 5 min. After rinsing the slides with a gentle stream of water, 25% sulphuric acid was used to de-colorize the smears for 2-4 min and if necessary de-colorization step may be repeated for another 1-3 min. The slides were rinsed as above and counterstained with 0.1% methylene blue for 30 seconds. The slides were then washed, air dried and examined under oil immersion. 
Modified ZN staining
This is very similar to that of standard ZN staining, except that the primary staining using 1% CF was 15 min.
Grading of smear
The smears were graded using 100 × oil immersion objective as per the RNTCP technical manual. 
| ~ Results|| |
During the study period a total of 1605 patients were included. In spite of continuous motivation, 68 (4.2%) patients dropped out. The results of the remaining 1537 patients are given. SSP was 9.43% (146 patients) in SM scheme with standard ZN staining-whereas this was increased to 9.8% (151 patients) with modified ZN staining. Similarly for SS 2 approach, SSP was 9.37% (144 patients) and 9.8% (151 patients) with standard and modified ZN staining procedures, respectively [Table 1].
| ~ Discussion|| |
The main aim of our study is to see the utility of SS 2 for diagnosis of TB. The SS 2 approach initiates treatment on the same day. In developing countries like India, TB is a common community health hazard and majority of infected individuals usually are below poverty line. Nearly 90% of TB cases occur in middle- and low-income countries such as India, Pakistan, Africa.  In these developing countries ssm is the only diagnostic method to detect TB, as the people can ill-afford for rapid TB diagnostic tests. In our study also, 43% of the patients income is less than rupees 3200 per month. Most of our patients were daily labourers.
In our study, majority of patients refused to come second time to submit M sample. In the study by Cuevas et al.,  patients were asked to submit three sputum samples by taking ssm0 or SMS scheme. Patients who were on ssm0 scheme submitted more number of first two sputum samples.
In a study by Van Deun et al.,  SSP was 11.7% when smears are exposed to primary staining step for 15 min. With standard ZN staining the SSP was 11.1%. In our study SSP was same (9.8%) for both the approaches with modified ZN staining. When standard ZN staining was used, the SSP was 9.43% for SM approach and 9.37% for SS 2 approach. Statistically there was no significant difference between the two schemes (P > 0.05). But two cases were missed, which is against global target for TB control.
The practical difficulty in our study was rejection of spot sample results by the clinicians due to 10% higher sensitivity of single morning sample.  But our study proved that two spot sample results were equally good compared with morning sample. Due to high bacillary load more number of samples showed 3 + positive result in SM approach: 34 and 20, respectively, when modified and standard ZN staining procedures were used [Table 2]. Whereas 3 + positivity was just 31 and 19 with SS 2 approach for the two ZN (modified and standard) staining procedures, respectively [Table 3].
Patient dropout was 6.3% in our previous study on SS 2 .  In the current study also extra efforts were put to motivate the patients. Despite this, patient dropouts were 4.2% in the current study. In field conditions, this number could definitely be much more. ,, The global target for TB control is to detect 70% smear positive cases. It was reported in WHO 2002 bulletin that this goal will not be reached until 2013.  According to Cambanis et al.,  TB control measures are compromised by lack of inexpensive, sensitive and robust diagnostic tests. However, patient-dropout should be considered foremost in this list.
| ~ Conclusion|| |
conclude, ZN staining results of SS 2 approach are very similar to SM approach and dropout rate is nil because once the patient visits a hospital or health care setup, few hours waiting is acceptable rather than coming for a second visit. Hence the TB control programmes need to consider revising the norms of sputum collection schemes to SS 2 in place of SM approach with prolonged primary staining step. This may contribute to global TB control rather than ssm alone.
| ~ References|| |
|1.||Bonnet M, Ramsay A, Gagnidze L, Githui W, Gurin PJ, Varaine F. Reducing the number of sputum samples examined and thresholds for positivity: An opportunity to optimize smear microscopy. Int J Tuber Lung Dis 2007;11:953-8. |
|2.||Cambians A, Ramsay A, Wirkom V, Tata E, L Cuevas E. Investing time in microscopy: An opportunity to optimize smear based case detection of tuberculosis. Int J Tuber Lung Dis 2007;11:40-5. |
|3.||Squire SB, Belaye AK, Kashoti A, Salaniponi FM, Mundy CJ, Theobald S, et al. Lost smear positive pulmonary tuberculosis cases: Where are they and why did we loose them? Int J Tuber Lung Dis 2005;9:25-31. |
|4.||WHO 2007 annual report. Available from: www.who.int/entity/whr/2007/whr07en.pdf [Last accessed on 2012 Dec 16]. |
|5.||Chandra TJ. Same day sputum smear microscopy approach for the diagnosis of pulmonary tuberculosis in a microscopy center at Rajahmundry. Indian J Tuberc 2012;59:141-4. |
|6.||RNTCP Central TB Division. Manual for Laboratory Technicians. New Delhi, India: Directorate General of Health Services, Ministry of Health and Family Welfare, 1998. Available from: http://www.tbcindia.org/LABMANUAL.pdf [Last accessed date on 2013 Jan 22]. |
|7.||Selvakumar N, Gomathi Sekar M, Rahman F, Syamsunder A, Duraipandian M, Wares F, et al. Comparison of variants of carbol-fuchsin solution in Ziehl-Neelsen for detection of acid-fast bacilli. Int J Tuberc Lung Dis 2005;9:226-9. |
|8.||Selvakumar N, Rahman F, Rajasekaran S, Narayanan PR, Frieden TR. Inefficiency of 0.3% Carbol Fuchsin in Ziehl-Neelsen Staining for Detecting Acid-Fast Bacilli. J Clin Microb 2002;40:3041-3. |
|9.||Angra P, Ridderhof J, Smithwick R. Comparison of two different strengths of Carbol Fuchsin in Ziehl Neelsen staining for detection Acid Fast Bacilli. J Clin Microb 2003;7:3459. |
|10.||Weyer K. Laboratory services in tuberculosis control. Part II. Microscopy. World Health Organization Technical Bulletin 98. 258. Geneva, Switzerland: WHO; 1998. |
|11.||International union against tuberculosis and lung disease. Sputum examination for tuberculosis by direct microscopy in low-income countries. Technical Guide. 5 th ed. Paris: IUATLD; 2000. |
|12.||Van Deun A, Hamid Salmin A, Aung KJ, Performance of variations of carbolfuchsin staining of sputum smears for AFB under filed conditions. Int J Tuberc Lung Dis 2005;9:1127-33. |
|13.||World Health Organization (2009) Global tuberculosis control: epidemiology, strategy, financing. Geneva. Available from: www.who.int/tb/publications/global-report/2009/pdf/full-report.pdf [Last accessed on 2012 Dec 10]. |
|14.||Cuevas LE, Yassin MA, Al Sonboli N, Lawson L, Arbide I, Al-Aghbari N, et al. A multi country non inferiority cluster randomized trial of frontloaded smear microscopy for the diagnosis of pulmonary tuberculosis. PLoS Med 2011;8:e1000443. |
|15.||Cuevas LE, Al-Sonboli N, Lawson L, Yassin MA, Arbide I, Al-Aghbari N, et al. LED fluorescence microscopy for the diagnosis of pulmonary tuberculosis: A multi-country cross-sectional evaluation. PLoS Med 2011;8:e1001057. |
|16.||Chandrasekaran V, Ramachandran R, Cunningham J, Balasubramanium R, Thomas A, Factors leading to tuberculosis diagnostic drop-out and delayed treatment initiation in Chennai, India. Int J Tuber Lung Dis 2005;9:172. |
|17.||Keeler E, Perkins MD, Small P, Hanson C, Reed S, Cunningham J, et al. Reducing the global burden of tuberculosis cases: The contribution of improved diagnostics. Nature 2006;444:49-57. |
|18.||Dye C, Watt CJ, Bleed D. Low access to a highly effective therapy: A challenge for international tuberculosis control. Bull World Health Organ 2002;80:437-44. |
|19.||Cambanis A, Yassin MA, Ramsay A, Squire SB, Arbide I, Cuevas LE. A one day method for the diagnosis of pulmonary tuberculosis in rural Ethiopia. Int J TubercLung Dis 2006;10:230-2. |
[Table 1], [Table 2], [Table 3]