174 Review Article

BLEACH OPTIMIZATION OF SPUTUM SMEAR MICROSCOPY FOR PULMONARY TUBERCULOSIS

Padma Srikanth1, S. Kamesh2 and Peter Daley 3 (Received on 25.4.2009; Accepted after revision on 20.8.2009)
Summary: The Revised National Tuberculosis Control Programme (RNTCP) aims to improve case detection rates of tuberculosis to facilitate prompt recognition and treatment. The low case detection rates in the programme can be directly attributed to failure to screen patients with suspected tuberculosis and the low sensitivity of the direct smear microscopy method to detect cases among the fraction of patients that are screened. Apart from low sensitivity, this method also has other disadvantages including the increased risk of infection transmission to technicians. There are several methods that can be used to improve sensitivity, but their applicability in a national programme and in resource limited settings are limited. Bleach processing of sputum smears prior to microscopy may be a cheap and effective way to improve on the sensitivity of the direct smear. Four distinctive techniques of sputum smear processing using bleach are described in the review, with the variations in each technique, along with the sensitivity. An analysis of reports published earlier on the bleach method is also presented including a discussion on when and why the bleach method works.

[Indian J Tuberc 2009; 56:174-184 ] Key words: Tuberculosis, Bleach Sedimentation, Concentration Methods, Sputum Microscopy.

INTRODUCTION Tuberculosis claims over two million lives and causes an economic drain of over three billion rupees a year in India1-3 . The success rates of properly applied DOTS (directly observed treatment, short course) exceeds 80-85 % in most countries of south east Asia4, however the primary obstacle to complete control over the spread and incidence of the disease is poor case finding, which still remains much less than 70 % in some south east Asian countries such as India (61.3 %)4. When more cases are identified, more cases are cured and less transmission occurs, reducing the incidence of tuberculosis.5 Case detection is currently done using direct microscopy on three sputum samples, according to the recommendations of the Revised National Tuberculosis Control Programme (RNTCP). This method is known to have a sensitivity of 50-70 % 6,7. The United Nations Millennium Development Goals (MDGs) suggest that

to halve the prevalence rate by 2015, TB control programmes must reach global targets for detection (70%) and treatment success (85%), this is also the target of the RNTCP8. The Global Plan to Stop TB 2006-2015 recognizes the limitations of sputum smear direct microscopy and recognizes the need for research into new tools that will improve diagnostic detection of M. tuberculosis and shorten the time to diagnosis9. There are several other methods that can be used to improve sensitivity of detection of M. tuberculosis such as culture (LJ, MGIT, other liquid media). But these methods are limited by a long processing time and high cost. Newer molecular techniques like PCR, though rapid, are too expensive to be widely applied in resource limited settings. An alternative to culture is optimization of the sputum smear process. Sputum concentration methods like the traditionally used oxalic acid,

1. Professor, Department of Microbiology, Sri Ramachandra Medical College & Research Institute, Sri Ramachandra University, Porur, Chennai. 2. Intern, Sri Ramachandra Medical College & Research Institute, Sri Ramachandra University, Porur, Chennai. 3. Lecturer, Department of Medicine and IDTRC, Christian Medical College and Hospital, Vellore. Correspondence: Dr. Padma Srikanth, Department of Microbiology, Sri Ramachandra Medical College & Research Institute, Sri Ramachandra University, #1, Ramachandra Nagar, Porur, Chennai 600 116; Tel : +91-44-24765512 extn 230/258; Mobile : 9884389682; Fax : +91-44-24765995; e-mail : srikanth_padma@rediffmail.com

Indian Journal of Tuberculosis

PADMA SRIKANTH ET AL

175

sulphuric acid and sodium hydroxide, N-acetyl-Ncysteine-NaOH (NALC-NaOH) methods, and newer methods such as Phas (phenol ammonium sulphate) method, CB-18 (carboxypropylbetaine), Zephirantrisodium phosphate method (Z-TSP), chitin sedimentation method, are available. The Phas method yields aesthetically appealing, better adhering smears but with a low increase in sensitivity (2 %)10,11. CB-18 method is both non-specific and expensive and does not increase sensitivity by much when compared to NaLC-NaOH method (61.9 to 71.4 %)8,12-14. Chitin provides greater sensitivity than bleach (83 % compared to 79 %) in a shorter period of digestion but remains more expensive than bleach and much less readily available to enable widespread application15. This review discusses the different optimization methods using bleach, the advantages and disadvantages, and presents in detail the methods of optimization and factors affecting the applicability of the bleach method. The standard way to assess the superiority of a particular method is by measuring its incremental yield over direct smear, or by the sensitivity of the methods when compared to culture. The best method for case detection for tuberculosis must be a method that has a high case detection, is easy to use, preferably not much variant from the regular techniques, should be applicable on a large scale and most importantly must be costeffective. Bleach may be one such method. Steingart and Angeby in two elegant reviews on the use of various methods to improve case detection by sputum microscopy demonstrated the effectiveness of bleach16,17. Bleach method a historical perspective The use of bleach can be traced back to 1909, where sodium hypochlorite (bleach) was used as a mucolytic before centrifuging sputum samples26. This involved the addition of bleach to the sputum sample before the sample was subjected to centrifugation and then processed by direct microscopy after staining with the Zeihl Neelsen method. The bleach method has undergone several modifications and diversifications and has recently

received more attention with the advent of the HIV epidemic. After a review of articles published on the bleach method, four distinctive methods for sputum processing with bleach can be identified. These methods are shown in Figure 1 and include 1. a method using centrifugation.(Figure 1a) 2. a method using overnight sedimentation27, (Figure 1b) 3. a method where a timed smear is used (Figure1c) 4. a method where a mixed smear (bleach processed (overnight) smear is added on top of a direct smear) is used28. METHODS Method using centrifugation This method was initially described by Gebre et al29 in 1995 and subsequently used as is, or as a modified technique that used centrifugation for sputum processing (method A figure 1). Each sample is treated as a separate sample (meaning that all samples from a single patient are not mixed together for assessment) and an approximately equal amount of sodium hypochlorite is added to the sputum sample. The sample is then incubated at room temperature for 15 min and shaken regularly to ensure homogenization. After 15 min, double the amount of the original sample volume of distilled water is added and the sample is then centrifuged in an appropriate centrifuge (1500-4000 rpm) for a duration averaging at 20 min. Post centrifugation, a drop of the sediment is then smeared onto a labelled slide using a pipette. As a variation of the method discussed above, eight ml of distilled water can be added to the sample irrespective of the sample volume20,21, also the rate of centrifugation used, may vary. Method using overnight sedimentation An approximately equal amount of sodium hypochlorite (usually 5 % here) is added to the

Indian Journal of Tuberculosis

176

OPTIMAL BLEACH METHOD FOR PULMONARY TB DETECTION

Figure 1 : Methods of bleach sedimentation for sputum smears

Indian Journal of Tuberculosis

PADMA SRIKANTH ET AL

177

sample and is left at room temperature overnight; the duration of sedimentation is usually more than 12 h (method B figure 1). Steingart analysed the bleach sedimentation methods as those that have sedimentation time less than or greater than one hour. The amount of time taken for sedimentation in the more than one hour category can vary between 12 - 24 h. The supernatant is discarded and the sediment is mixed with the remaining fluid and smeared onto a labelled slide. Variations of the above method include the addition of distilled water before the process of overnight sedimentation28 and the use of different concentrations of sodium hypochlorite. Method using a 30-45 minute timed smear Samples are labelled appropriately and approximately equal volume of sodium hypochlorite is added (method C figure 1). The sample container is left on a laboratory ledge at an angle of 45 degrees (to facilitate sedimentation) for duration of 30-45 min, and the sediment is smeared onto a labelled slide. Method using a mixed smear The sample is allowed to undergo autodigestion overnight following which a thick smear is made (method D figure 1). This auto-digested sample is then processed by the overnight bleach sedimentation method and another smear is made on the previously made thick smear on a labelled slide. FINDINGS Effect of bleach pre-treatment The summary of the studies assessed by this review, are shown in Table 1 and indicate the sample size, the comparative method, the grading used and their results. Some studies report sensitivity as high as 92.3 % using the bleach method when compared to the WHO case definition19. This is a substantial increase from the usual 50-70 % that the direct smear microscopy method provides.

Bleach method30 allows for detection of samples that were negative by the direct method, 12 of 42 samples positive by this method (29 %) were negative by the direct method. However, the overall sensitivity did not improve since an equal number of samples that were positive using the direct smear method were negative using the bleach method (13 of 42 (31 %) positive by the direct method). The authors went on to suggest that to improve sensitivity, samples that were negative by the direct smear method be reprocessed with bleach. An increase in sensitivity with bleach was also documented in another study28, however this was limited to patients with a low sputum bacillary load (sensitivity increased while using the American Thoracic Society (ATS) grades instead of the International Union Against Tuberculosis and Lung Disease (IUATLD) grades - The ATS scale considers less than ten bacilli to be positive and defines it as scanty while the IUATLD scale does not consider this significant). They also reported an increase in sensitivity (by around one percent in a large sample size of around 3500 patients) when patient detection instead of smear positivity, was used as the criteria, and went on to suggest that the increase in sensitivity while using bleach was actually due to false positives. However, a review of the methodology used in the study showed that culture was not used as a gold standard. Therefore, the claims of false positivity have to be viewed critically. Fourteen studies described methods that used centrifugation during processing of sputum. However, even though all these studies did use centrifugation, they had minor variations in the exact method used. The variations were confined to the amount of water added and speed during centrifugation and may or may not account for the range of sensitivity observed by these studies. Of the five studies that described methods that processed sputum by the overnight sedimentation method, the average increase in sensitivity was at 5.39 %, the highest being 17 % and the lowest at one percent. These studies vary in

Indian Journal of Tuberculosis

178

OPTIMAL BLEACH METHOD FOR PULMONARY TB DETECTION

Indian Journal of Tuberculosis

PADMA SRIKANTH ET AL

179

Indian Journal of Tuberculosis

180

OPTIMAL BLEACH METHOD FOR PULMONARY TB DETECTION

their exact duration of sedimentation and also possess minor variations in the method used. Before coming to a conclusion that centrifugation is the better method when compared to overnight sedimentation, operational considerations such as cost, technical and feasibility factors which exist in the installing of a centrifuge to peripheral laboratories, and issues such as frequent power cuts or power failures in resource limited settings must be considered. The risk of exposure to bio-hazardous material is also a concern while using centrifugation. Moreover, overnight sedimentation method does produce a good increase in sensitivity and is free of the hassles of the use of a centrifuge. Effect of bleach pre-treatment in HIV setting There are few studies on use of bleach to improve detection among HIV infected individuals. Reports suggest that this method improves the sensitivity of the smears from 39 - 50 % in a large cohort of patients from Ethiopia31. A study using bleach and centrifugation reported increase in sensitivity by 11 %31,32. A community based study33 with high prevalence of HIV (over 50 %) reported a significant improvement in smear microscopy using bleach sedimentation and concluded that the increased sensitivity remained significant even when a sensitive AFB threshold was used, such as the ATS scale which considers even 1-10 bacilli in all fields screened to be positive for tuberculosis. A study evaluating bleach concentrated fluorescent microscopy smears in the diagnosis of tuberculosis in the HIV infected, documented an increase in yield of 14.9 % and 17.2 % in culture positive and culture negative HIV patients respectively57. A study 59 using bleach for smear microscopy in HIV positive patients stated that bleach processing had similar recovery rates in both HIV positive and negative individuals in definite cases defined as two or more positive smears and three per cent in very likely cases defined as smearnegative, culture- positive tuberculosis. Another study on selectively using the bleach method in smear negative patients found that bleach can

considerably improve the sensitivity of acid fast staining (sensitivity of 27.1 % in previously smear negative patients when compared to culture) 60. Patients with HIV have a low sputum bacillary load which requires a more sensitive method for case detection.61 Advantages of bleach pre-treatment Bleach is cheap, readily available and improves the sensitivity of the direct microscopy method considerably. Bleach is also an effective disinfectant, and therefore helps reduce the infection risk to laboratory personnel. Most other methods that claim to improve case detection require biosafety cabinet level II or higher, which are not available in the peripheral laboratories and are not cost-effective in field conditions. Moreover, bleach can also be conveniently added to the sputum container directly, as the first step in the processing of the sample, reducing the chances of infection during sample transfer and processing. Githui et al specifically assessed this property of bleach in circumstances where it is used as a concentrating agent and found it to be effective at killing tuberculous bacillus34,35. Some researchers have tried the bleach method on extra-pulmonary tuberculosis. Samples such as abscess fluids, lymph node aspirates, body fluids or skin scrapes and found the method and documented that bleach did improve sensitivity here too22,56. The sensitivity reported was 43.4 % on cytology, 21.8 % on direct ZN, 70.9 % on bleach microscopy among 55 samples processed (22). Previous studies 18,35,36 suggest that the sensitivity with the bleach method is so high that it may be possible to reduce the number of sputum samples required for the diagnosis of tuberculosis from three to just one. This would mean that only the first on the spot sample is used considerably reducing the workload in busy laboratories. It is already known that the increase in detection with the third sputum sample is only 2 5 %, meaning that the collection of three samples is unnecessary. Collection of a single sample may improve patient compliance and reduce cost.

Indian Journal of Tuberculosis

PADMA SRIKANTH ET AL

181

Disadvantages of bleach pre-treatment Bleach interferes with the enzymatic processes of mycobacteria owing to the oxidative effects of chlorine and hence is an effective disinfectant. But this also means that bleach processing of a sample of sputum is an end point, as it cannot be further subjected to culture. Hypochlorite loses its concentration with time with a decline in free chloride concentration of 2-5 % at the end of 60 days 40 . The sodium hypochlorite molecule undergoes spontaneous thermal decomposition to form either chlorate (dominant) or oxygen in solution. If a specific concentration of hypochlorite is required for effectivity of the method, then this factor must be considered prior to its application in the field. There is also no definitive consensus on the concentration or duration of bleach to be used, though 5 % hypochlorite is most frequently used as it is commercially available as 5 6 % sodium hypochlorite. Effectiveness of the method has been shown with concentrations as low as 3.5 %18 and one percent41. Another possible disadvantage with the bleach method is that it seems to be of a low sensitivity when sputum volume is low42. It may not show greater sensitivity than the direct method when all other factors influencing the smear results are optimized. Optimizing the smear collection methods through patient education and supervision, early and appropriate processing of sputum smears by trained and skilled lab assistants, proper reading of smears for a minimum of 5-10 min by two independent observers and a good quality control system seemed to improve sensitivity considerably, and bleach may not be able to improve sensitivity any further. However optimizing all these factors may not be feasible within the national TB control program in India. Bleach smears also have practical problems in being more fragile and being harder to identify on a slide because bleach digests the proteins that help the smear to stick on to the slide18.

If centrifugation were important to increase sensitivity by the bleach method, its applicability in resource poor settings would be considerably reduced. Centrifugation only seems to improve the grade of the sputum specimen and does not affect the sensitivity according to a study in Ethiopia29. However, the highest positivity rates of smears processed by centrifugation on average remain higher than overnight sedimentation. The increase in sensitivity using centrifugation over overnight sedimentation must be reviewed with the cost of centrifugation taken into account in resource limited settings. A study assessing the sensitivity of the timed smear method at various times of bleach sedimentation from 15 - 60 min, found that the best results were obtained when the processing time was 30 - 45 min, which also means that if sedimentation were to take place for say 60 min, the sensitivity is reduced18,19. The delay in providing results when using methods involving overnight sedimentation is a cause for concern. RNTCP mandates use of three samples for screening, WHO advocates two samples as being sufficient, because it is the first two samples that contribute to sensitivity. This implies that the probability that a patient may be first detected to be AFB positive on direct microscopy of the third sample when the first two samples have been negative is negligible42-49. Studies suggest that by the use of bleach, these two samples can be reduced to one with the single sample providing an improved sensitivity over the three samples. Why does it work? Bleach may help concentrating the sputum sample, especially when the sputum is processed by the overnight sedimentation method, where it is considered as a substitute to centrifugation. Others have attributed the improvement of sensitivity to the property of bleach to digest the debris (including mucous, salivary particles and cells) and help easily identify the bacteria in a much clearer field19,21,34.

Indian Journal of Tuberculosis

182

OPTIMAL BLEACH METHOD FOR PULMONARY TB DETECTION

One other important point of view regarding the success of the bleach method is with reference to the fact that the method may show an improved sensitivity in a study simply because the ATS scale was used instead of the IUATLD scale28,38,39. This means that the bleach method helps in improving the sensitivity only when the bacterial load is low (<10 bacilli per field) and therefore is more useful in the HIV infected. Bleach and fluorescent microscopy New innovative techniques to use bleach with fluorescent microscopy are now being tested at some centres 57,58. However, the cost and availability of fluorescent microscopes at peripheral areas is a limiting factor for the application of this method. CONCLUSION The limits of the bleach method have been tested considerably over the last decade. It has proved to be an effective method to improve the sensitivity of case detection. It is a safe, cheap, applicable, easy to use and effective method applicable even in patients with low sputum bacillary load, such as those with HIV. The sedimentation method can be used in resource poor settings to improve sputum smear sensitivity considerably. An expert consultation group aimed at improving the diagnosis of tuberculosis through the optimisation of sputum microscopy suggested that multi-centre studies be conducted to determine the sensitivity and specificity of a revised case definition with one positive smear result, prospectively evaluate the relative yield of serial sputum specimens, and define the optimal timing of specimen collection that minimizes delay in the diagnostic pathway. It also suggested an internationally co-ordinated and standardised approach to strengthen the country specific evidence base and permit comparison54. However, some of the issues which need to be addressed before a large scale implementation include decisions on which one of the described methods are to be applied, what concentration of

bleach are to be used, how many sputum samples are required for diagnosis when the bleach method is followed, does the loss of concentration of sodium hypochlorite with time affect the process and if centrifugation is dispensable considering the cost factor. The WHO is currently assessing the applicability of bleach in several countries and the national programme must look out for results of these studies and seriously consider bleach as a possible aid for the diagnosis of tuberculosis in India. This review has presented a composite picture of different processes used in bleach which would aid optimization in its application in smear microscopy for pulmonary tuberculosis. REFERENCES
WHO, Global tuberculosis programme, Global tuberculosis Control, World Report 1999, Geneva: WHO/CDS/CPC/ TB/90-259. 2. Murray CR, Styblo K, Rouillon. Tuberculosis in develoing countries: burden, intervention and cost. Bull Int Union Tuberc Lung Dis 1990; 65:1-20. 3. RNTCP, Technical and operational guidelines for tuberculosis control, October 2005: 1-4. 4. http://www.who.int/whosis/database/core/core_select.cfm 5. Christopher Dye, Geoffrey P Garnett, Karen Sleeman, Brian G Williams Prospects for worldwide tuberculosis control under the WHO DOTS strategy. Lancet 1998; 352 : 1886-91. 6. Case-detection and treatment success rates in RNTCP areas for 2003/2004, Published by the RNTCP, India. 7. VK Dhingra, Nishi Aggarwal, S Rajpal, JK Aggarwal, SN Gaur Validity and Reliability of Sputum Smear Examination as Diagnostic and Screening Test for Tuberculosis. Indian J Allergy Asthma Immunol 2003; 17 : 67-69. 8. Elizabeth L. Corbett; Catherine J. Watt; Neff Walker; Dermot Maher; Brian G. Williams; Mario C. Raviglione; Christopher Dye. The Growing Burden of Tuberculosis, Global Trends and Interactions with the HIV Epidemic. Arch Intern Med May 12 2003, Vol 163. 9. Mario C Raviglionea. The new Stop TB Strategy and the Global Plan to Stop TB, 20062015. Bulletin of the World Health Organization 2007; 85. 10. N. Selvakumar, Fathima Rahman, Renu Garg, S. Rajasekaran, Nalini Sunder Mohan, K. Thyagarajan, V. Sundaram, T. Santha, Thomas R. Frieden, and P. R. Narayanan. Evaluation of the Phenol Ammonium Sulfate Sedimentation Smear Microscopy Method for Diagnosis of Pulmonary Tuberculosis. Journal of Clinical Microbiology. 2002; 40: 301720. 11. Collin CH, JM Grange, MD Wates. Tuberculosis bacteriology: Organization and practice (2nd edn). Oxford: Butterworth-Heinemann, Cambridge UK, 1997. 1.

Indian Journal of Tuberculosis

PADMA SRIKANTH ET AL
12. Charles G. Thornton, Kerry M. Maclellan, Thomas Brink, Jr., Denise E. Lockwood Mark Romagnoli, June Turner, William G. Merz, Richard S. Schwalbe, Marcia Moody, Yvonne Lue, Selvin Passen. Novel Method for Processing Respiratory Specimens for Detection of Mycobacteria by Using C18-Carboxypropylbetaine: Blinded Study. Journal of Clinical Microbiology 1998; 36: 199603. 13. Cherise P. Scott, Luciano dos Anjos Filho, Fernanda Carvalho de Queiroz Mello, Charles G. Thornton, William R. Bishai, Leila S. Fonseca, Afranio L. Kritski, Richard E. Chaisson, and Yukari C. Manabe. Comparison of C18Carboxypropylbetaine and Standard N-Acetyl-L-Cysteine NaOH Processing of Respiratory Specimens for Increasing Tuberculosis Smear Sensitivity in Brazil. Journal of Clinical Microbiology 2002; 40: 321922. 14. K. F. Laserson, N. T. N. Yen, C. G. Thornton, V. T. C. Mai, W. Jones, D. Q. An, N. H. Phuoc, N. A. Trinh, D. T. C. Nhung, T. X. Lien,N. T. N. Lan, C. Wells, N. Binkin, M. Cetron, S. A. Maloney. Improved Sensitivity of Sputum Smear Microscopy after Processing Specimens with C18-Carboxypropylbetaine to Detect Acid-Fast Bacilli: a Study of United States-Bound Immigrants from Vietnam. Journal of Clinical Microbiology 2005; 43: 34603462. 15. P. Farnia, F. Mohammadi, Z. Zarifi, D. J. Tabatabee, J. Ganavi, K. Ghazisaeedi, P. K. Farnia, M. Gheydi, M. Bahadori, M. R. Masjedi, A. A. Velayati . Improving Sensitivity of Direct Microscopy for Detection of AcidFast Bacilli in Sputum: Use of Chitin in Mucus Digestion. Journal of Clinical Microbiology. 2002; 40: 508511. 16. Angeby KA, Hoffner SE, Diwan VK Should the bleach microscopy method be recommended for improved case detection of tuberculosis? Literature review and key person analysis. International Journal of tubercular Lung Diseases 2004; 8: 806-15. 17. Karen R Steingart, Vivienne Ng, Megan Henry, Philip C Hopewell, Andrew Ramsay, Jane Cunningham, Richard Urbanczik, Mark D Perkins, Mohamed Abdel Aziz, Madhukar Pai. Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 2006; 6: 66474. 18. Lawson L, Yassin MA, Ramsay 1, Olajide I, Thacher TD, Davies PD, Squire SB, Cuevas LE. Microbiological validation of smear microscopy after sputum digestion with bleach; 1 step closer to a one-stop diagnosis of pulmonary tuberculosis. Tuberculosis (Edinb) 2006; 86: 34-40. 19. Yassin MA, Cuevas LE, Gebrexabher H, Squire SB.: Efficacy and safety of short-term bleach digestion of sputum in case-finding for pulmonary tuberculosis in Ethiopia. Int J Tuberc Lung Dis 2003; 7: 678-83. 20. Miorner H, Ganlov G, Yohannes Z, Adane Y..Miorner. J Improved sensitivity of direct microscopy for acid-fast bacilli: sedimentation as an alternative to centrifugation for concentration of tubercle bacilli. Clin Microbiol 1996; 34: 3206-7. 21. Saxena S, Mathur M, Talwar VK.. Detection of tubercle bacilli in sputum: application of sodium hypochlorite concentration method J Commun Dis 2001; 33: 241-4.

183

22. Khubnani H, Munjal K Application of bleach method in diagnosis of extra-pulmonary tuberculosis.. Indian J Pathol Microbiol 2005; 48: 546-50. 23. Mutha 1, Tiwari S, Khubnani H, Mall S..Application of bleach method to improve sputum smear microscopy for the diagnosis of pulmonary tuberculosis. Indian J Pathol Microbiol 2005; 48: 513-7. 24. Kochhar A.Evaluation of a two step AFB cold staining method and simplified concentration technique for diagnosis of pulmonary tuberculosis. J Commun Dis 2002; 34: 276-80. 25. V. M. Katoch. Smear microscopy to diagnose tuberculosis. Indian J Med Res 2006; 123: 735-38. 26. Uhlenhut , Xylander Untersuchungen uber antiformin. Arb K Gesund 1909; 32: fasc. 1, 158. 27. Gebre-Selassie S Evaluation of the concentration sputum smear technique for the laboratory diagnosis of pulmonary tuberculosis. Trop Doct 2003; 33: 160-2. 28. Deun, Maug AK, Cooreman E, Hossain MA, Chambuganj N, Rema V, Marandi H, Kawria, Portaels F. Bleach sedimentation method for increased sensitivity of sputum smear microscopy: does it work? Int J Tuberc Lung Dis 2000; 4: 371-6. 29. Gebre N, Karlsson U, Jonsson G, Macaden R, Wolde, Assefa, Miorner H.. Improved microscopical diagnosis of pulmonary tuberculosis in developing countries. Trans R Soc Trop Med Hyg 1995; 89: 191-3. 30. Wilkinson D, Sturm AW. Diagnosing tuberculosis in a resource-poor setting: the value of sputum concentration. Trans R Soc Trop Med Hyg 1997; 91: 420-1. 31. Bruchfeld J, Aderaye G, Palme IB, Bjorvatn B, Kallenius G, Lindquist L. Sputum concentration improves diagnosis of tuberculosis in a setting with a high prevalence of HIV. Trans R Soc Trop Med Hyg 2000; 94: 677-80. 32. Angeby KA, Alvarado-Galvez C, Pineda-Garcia L, Hoffner SE. Improved sputum microscopy for a more sensitive diagnosis of pulmonary tuberculosis. Int J Tuberc Lung Dis 2000; 4: 684-7. 33. Maryline Bonnet, Andrew Ramsay, Willie Githui, Laramie Gagnidze, Francis Varaine, Philippe J. Guerin. Bleach Sedimentation: An Opportunity to Optimize Smear Microscopy for Tuberculosis Diagnosis in Settings of High Prevalence of HIV. Clinical Infectious Diseases 2008; 46: 171016. 34. Githui WA, Matu SW, Tunge N, Juma E. Biocidal effect of bleach on Mycobacterium tuberculosis: a safety measure. Int J Tuberc Lung Dis 2007; 11: 798-802. 35. M. Best, S. Sattar, V. S. Springthorpe, M. E. Kennedy. Comparative Mycobactericidal Efficacy of Chemical Disinfectants in Suspension and Carrier Tests. Appl. Environ.Microbiol 5 4(11). 36. Mase SR, Ramsay A, Ng V et al. Yield of serial sputum examinations in the diagnosis of pulmonary tuberculosis: a systematic review. Int J Tuberc Lung Dis 2007; 11: 48595. 37. Allwood M, Lee Y, Salaniponi F, Nyangulu D, Kuppusamy I, Squire S. Case-finding with a single sputum sample and household bleach. Int J Tuberc Lung Dis 1997; 1 (suppl 1): S144.

Indian Journal of Tuberculosis

184

OPTIMAL BLEACH METHOD FOR PULMONARY TB DETECTION

51. Habeenzu C, Lubasi D, Fleming AF. Improved sensitivity of direct microscopy for detection of acid-fast bacilli in sputum in developing countries. Trans R Soc Trop Med Hyg 1998; 92: 415-6. 52. Douthwaite S, Yassin MA, Squire SB, Arbide I, Cuevas LE.Bleach-digested sputum smears for the diagnosis of TB in HIV-infected individuals.Trop Doct 2007; 37: 35-6. 53. Makunde WH, Makunde RA, Kamugisha LM, Mgema SG, Liwa A.Improved microscopy diagnosis of pulmonary tuberculosis using sodium hypochlorite concentration technique in Tanga, Tanzania.Tanzan Health Res Bull 2007; 9: 87-93. 54. Aung WW, Nyein MM, Ti T, Maung W Improved method of direct microscopy for detection of acid-fast bacilli in sputum. Southeast Asian J Trop Med Public Health 2001; 32: 390-3. 55. Mohamed A Aziz, ;Luis E. Cuevas, Jane Cunningham, Adalbert Laszlo, Sundari Mase, Madhukar Pai,; Mark D; Krystyna Ryszewska,, S. BertelSquire,; Karen Steingart,;Veronique Vincent,; Karin Weyer,; Mohammed A. Yassin,: John Ridderhof. Improving the diagnosis of tuberculosis by the optimization of sputum microscopy. Expert consulatation, WHO headquarters, Geneva. 56. Gangane N, Anshu , Singh R. Role of modified bleach method in staining of acid-fast bacilli in lymph node aspirates. Acta Cytol 2008; 52: 325-8. 57. Eyangoh SI, Torrea G, Tejiokem MC, Kamdem Y, Piam FF, Noeske J, Van Deun A. HIV-related incremental yield of bleach sputum concentration and fluorescence technique for the microscopic detection of tuberculosis. Eur J Clin Microbiol Infect Dis 2008; 27: 849-55. 58. Matu S, Githui WA, Muthami LN, Makumi J, Ngeranwa J, Tunge N, Juma E, Karimi F. Combination of bleach and flourescent microscopy: a milestone in the diagnosis of smear negative tuberculosis. East Afr Med J 2007; 84: 460-5. 59. Lawson L, Yassin MA, Ramsay A, Emenyonu EN, Thacher TD, Davies PD, Squire SB, Cuevas LE. Short-term bleach digestion of sputum in the diagnosis of pulmonary tuberculosis in patients co-infected with HIV. Tuberculosis (Edinb) 2007; 87: 368-72. 60. Githui WA, Matu SW, Muthami LN, Juma E. Improved diagnosis of Ziehl-Neelsen smear negative tuberculosis using sodium hypochlorite sedimentation method. East Afr Med J 2007; 84: 455-9. 61. Elliot AM, Namaato K, Allen BW, Luo N, Hayls RJ, Pobee JO, McAdam KP. Negative sputum smear results in HIV positive patients in pulmonary tuberculosis in Lusaka, Zambia. Tuber Lung Dis 1993; 74: 191-94.

38. Ramsay, Squire SB, Siddiqi K, Cunningham J, Perkins MD.Bleach microscopy method and case detection for tuberculosis control. Int J Tuberc Lung Dis 2006; 10: 256-8. 39. A. Van Deun, A. Hamid Salim, E. Cooreman, P. Daru, A. P. K. Das, K. J. M. Aung, H. L. RiederScanty AFB smears: whats in a name? Int J Tubercul lung dis 2004; 8: 816 23. 40. Dj Pecora,DMZ Geurisoli, R S de silva, L P Unpublished observation. Shelf life of sodium hypochlorite. Vansan University of Saint Paul, Portugal. Available at (forp.usp.pr/ restauroda/soda/sodaingl.html) 41. Frimpong EH, Adukpo R, Owusu-Darko K. Evaluation of two novel Ziehl-Neelsen methods for tuberculosis diagnosis. West Afr J Med 2005; 24: 316-20. 42. Garay JE Analysis of a simplified concentration sputum smear technique for pulmonary tuberculosis diagnosis in rural hospitals.Trop Doct 2000; 30: 70-2. 43. Mabaera B,Naranbat N, Dhlivayo P, Reider HL. Efficacy of serial smear examinations in excluding sputum smear positive tuberculosis. Int J Tubercul Lung Dis 2006; 10: 1030-5. 44. Leonard MK, Osterholt D, Kourbatova EV, Del Rio C, Wang W, Blumberg HM. How Many sputum smears are required to diagnose pulmonary tuberculosis? Am J Infect control 2005; 33: 58-61. 45. Gopi PG,subramani R, Selvakumar N, Santha T, Eusuff SI, Narayanan PR. Smear examination of two specimens for diagnosis of pulmonary tuberculosis in Thiruvallur district,South India. Int J Tubercul Lung Dis 2004; 8: 824-8. 46. Yassin MA, Cuevas LE. How many sputum smears are necessary for case finding in pulmonary tuberculosis? Trop Med Int Health 2003; 8: 927-32. 47. Van deun A, Salim AH, Cooreman E, Hossain MA, Rema A, Chambugonj N, Hye MA, Kawria A, Declercq E. Optimal tuberculosis case detection by direct sputum smear microscopy : How much better is more? Int J Tubercul Lung Dis 2002; 6: 222-30. 48. Crampin AC, Floyd S, Mwangulu F, Black G, Ndhlovu R, Mwaiyeghele E, Glynn JR, Warndorff DK, Fine PE. Comparison of two versus three smears in identifying culture positive tuberculosis patients in a rural African setting with High HIV prevalence. Int J Tubercul Lung Dis 2001; 5: 994-9. 49. Wu ZL, Wang AQ. Diagnostic yield of repeated smear microscopy examinations among patients suspected of pulmonary TB in Shadong province in China. Int J Tubercul Lung Dis 2000; 4: 1086-87. 50. Harries AD, Mphasa NB, Mundy C, Banerjee A Kwanjana JH, Salaniponi FM. Screening tuberculosis suspects using two sputum Smears. Int J Tubercul Lung Dis 2000; 4: 36-40.

Indian Journal of Tuberculosis