Clorox Concentration Technique for the Demonstration of Acid Fast Bacilli in the Sputum Romulo Uy, M.D.,** Charles Yu, M.D.,** Mario Juco, M.D.,** Cora Adlawan, M.D.,** Grace Ruiz, M.D.,** Melecia Velmonte, M.D.** and Calixto Zaldivar, M.D.** (1st Prize, PSMID- Hoechst Annual Research Contest, 1987. UP-PGH Medical Center) ABSTRACT In countries like the Philippines where the prevalence of tuberculosis isi still high and here culture facilities are oftentimes unavailable, the identification of the tubercle bacilli in the sputum by microscopy is still the most accepted method for screening and follow-up of TB patients. Efforts therefore have to be geared toward improving yield of the bacilli in the sputum. In the Philippine General Hospital, the recovery of the acid-fast bacilli in the sputum was relatively low and concentration methods have been utilized to improve yield. This study was undertaken to compare the yield of TB bacilli by the usual direct method against two concentration techniques, which used sodium hypochlorite (Clorox) and sputufluol. A total of 297 sputum specimens were collected from 101 subjects with clinical and radiologic evidence highly suggestive of pulmonary tuberculosis. The mean age of these patients was 43.6 with a range from 17-76 years. The results showed that the direct smear had a positive yield of 35.4% while the concentration methods revealed a higher percentage, 45.8% for clorox and 46.1% for sputufluol. The difference was of statistical significance with a p value of < 0.001; there was a lesser degree of concordance between the two methods. In addition, there was no statistical significance between the two concentration methods, clorox versus sputufluol, with a p value of ≥ 0.05. The degree of concordance was high between the two methods. Thus, concentration methods were superior to the direct method in the recovery of the tubercle bacilli in the sputum. Clorox likewise can be a suitable alternative to sputufluol, an imported agent used in this hospital. In epidemiological case finding activities, it can replace the direct method to increase the yield of AFB in the smear. [Phil J Microbiol Infect Dis 1988; 17(1):13-18] Key Words: sodium hypochlorite, TB, AFB smear, tuberculosis INTRODUCTION Tuberculosis (TB) persists as an important health problem particularly in developing countries like the Philippines. According to the WHO figures, this country has the highest mortality rate worldwide 1 (Figure 1). Based on the 1987 survey of the Department of Health, although there was a decline in the mortality and morbidity of TB, it is the third leading cause of death in this country with a rate of 55 per 100,000 and ranks fifth in the leading causes of morbidity with a rate of 233.12 (Figure 2).In countries where the prevalence rate for tuberculosis is high, the sputum stained smear for acid-fast bacilli is the major epidemiological tool in the diagnosis of pulmonary tuberculosis. Smear positive tuberculous patients are given the highest priority in case finding and treatment since these patients are the most dangerous sources of infection. 3 The direct smear for AFB can detect one acid-fast bacillus per field when a milliliter of sputum contains at least 1,000,000 tubercle bacilli assuming an even distribution of acid-fast bacilli in the specimen and smear.3 Thus, it would be better to concentrate the organisms in the sputum. As early as 1915, Petroff 4 reported that Mycobacterium tuberculosis survived exposure to 4 percent sodium hydroxide for 24 hours, whereas other organisms were killed. In the next decade, newer agents were introduced. Krasnow and Wayne 4 reported that 3% sodium hydroxide was actually toxic to tubercle bacilli and recommended the use of trisodium phosphate. Krasnow and associates introduced zephiran into the trisodium phosphate, the former as a major decontaminating agent and the latter serving to liquefy sputum. 5 Other techniques utilized the phenol-autoclave concentration,6 N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method of Kubica and associates,7 and Clorox concentration method by Oliver and Reuser.8 Clorox contains 5.25% sodium hypochlorite as its active ingredient.8 Oliver and Reusser reported that it can concentrate more TB bacilli in the smear compared to other methods by digesting bulky sediments, pus, blood and mucus leaving a sediment consisting almost entirely of tubercle bacilli. These studies8,9 were done several years ago without statistical analysis. Since then, there has not been much interest regarding this reagent as a diagnostic tool for TB. Figure 2. Tuberculosis in the Philippines Figure 1: Tuberculosis mortality per 100,000 In the Philippine General Hospital the rate of recovery of acid-fast bacilli in thesputum is low (Table 1). The direct smear positivity rate (total positive smear/total number of specimens ranged from 4.5 to 10.6% (with a mean of 7.3%). For TB bacilli culture the PGH laboratory since 1985 has been using a special decontaminating agent imported from Germany (Sputufluol), the active ingredient of which is essentially sodium hypochlorite. The concentration of sodium hypochlorite in this reagent is relatively low (about 0.02%) compared to that of the local commercial version clorox (about 4.5%).These reagents were assayed in the Pharmacology Department of the University of the Philippines. Medline search since 1966 revealed no randomized well controlled trial had been done to confirm the 1942 report of Oliver and Reusser using clorox for concentration of TB bacilli sputum smears. Table 1. PGH data on direct smear and culture (1980-1986) Culture Results Positive Negative Total Direct Smear No. % No. % No % Positive 204 (62.2) 124 (37.8) 328 (7.3) Negative 32 (0.8) 4155 (99.2) 4187 (92.7) Total 236 (5.2) 4279 (94.8) 4515 (100.0) Source: Unpublished data by the authors This pilot study compares the yield of TB bacilli by the usual direct smear method against the two concentration methods employing sodium hypochlorite manufactured commercially as clorox, a locally available solution, and sputufluol, a reagent imported from Germany. MATERIALS AND METHODS A total of 101 patients were admitted to the study from November 1986 to July 1987. Patients with clinical and radiologic evidence highly suggestive .of active pulmonary tuberculosis and without previous intake of anti-TB medications for the past 3 months were included in the trial. On entry to the study, these patients were provided with sterile wide-mouthed .bottles and instructed on the proper way of collecting their sputum. Three consecutive early morning sputum specimens were collected amounting was to at least 10 ml each.10 Each sputum specimen was processed by three methods: the direct, clorox and sputufluol techniques (Figure3). A direct smear of each specimen collected was done immediately and the remain der was divided equally into two 50-ml screw-capped culture tubes. The tubes were labeled A and B respectively; tube A was used for the clorox mixture and Tube B for sputufluol method. For the direct smears a nickel-chrome wire loop was dipped into the sputum from the mixture in the wide-mouthed bottle. A smear was made onto a chemically clean slide. The most purulent, bloody and mucoid portion of the specimen was selected as recommended by different authors.11 Figure 3. Schematic diagram of the procedure of sputum specimen For the clorox concentration method the tube labeled A was placed on top of a serologic shaking machine and was shaken for 15 to 20 minutes. Clorox was added in a 1:1 dilution to the mixture after which it was centrifuged for 10 minutes at 3000 rpm. The supernatant liquid was discarded and the sediment was smeared onto a new, chemically clean slide by means of a nickel- chrome wire loop. For the sputufluol concentration method, the same procedure was done but sputufluol instead of clorox was used for Tube B. The sediments were spread over an area of 200 square mm (10 x 20 mm) and stained using the method of Kinyoun. 11 For cultures, the remaining sediments were neutralized with 1N HCI using phenolphthalein as indicator (solution turned colorless after the addition of the acid) and cultured onto Lowenstein-Jensen medium using the drop method. All the tubes were incubated at37°C and examined weekly for eight weeks for growth before it was finally discarded. All the slides were read by a qualified medical technologist who had no access to any information that might result in bias (another medical technologist prepared the slides). The reader reported the smears as positive or negative for acid-fast bacilli. Positive smears were further classified based on the American Thoracic Society scale 10,11,12 (Figure 4). A quantitative number was also specified by the reader based on the schema shown in Figure 4. As per recommendation by different authors, all slides were read under oil immersion with 3 1ongitudinal sweeps equivalent to about 300 fields. A minimum of 3 acid-fast bacilli per slide was required for the smear before it was considered as positive. Figure 4. Method of reporting of acid-fast bacilli in the smear as recommended by the American Thoracic Society Number of organisms seen Report 1. 0 in entire slide No AFB seen 2. 1-2 in entire slide Repeat collection 3. 3-9 per slide Rare 4. 10 or more per slide Few 5. More than one per OIF Numerous Chest x-rays were read officially by a senior radiologist and classified as to minimal, moderately advanced or far-advanced and whether a cavity was present or not.13 Patients with previous intake of anti-tuberculosis drugs for the past three months and those without cough were excluded from the study. The results were analyzed by the chi square test for paired samples specifically the Stuart Maxwell statistics at alpha = 0.05 to compare the direct smear versus the clorox method, direct versus sputufluol and clorox versus sputufluol techniques with respect to the number of acid-fast bacilli detected based on the American Thoracic Society criteria mentioned above. Kappa agreement and its 95% confidence interval were computed to determine further the agreement between the three methods. RESULTS A total of 297 sputum specimens were collected from 101 patients with a mean age of 43.5 years (17-76 years). There were 66 males and 35 females with a 1.9:1.0 ratio. The clinical characteristics of these patients are shown in Table 2. Table 2. Clinical profile of patients included in the study Number Percentage 1. Cough 101 100.0 2. Weight loss 62 61.4 3. Fever 53 53.5 4. Anorexia 49 49.0 5. Hemoptysis 45 45.0 The radiologic findings of these patients are shown in Table 3. Of the population under study, cavitary lesions in the chest x-ray were seen in 53 patients or 52.5% of 101 patients. Far advanced lesions comprised 45.5%, moderately advanced in 49.5% and minimal active lesions in 4.9% of all cases. All of the cases included in this study were classified as Class III based on the American thoracic Society classification of tuberculosis.14 Table 3. Radiologic interpretation of the chest x-rays Number Percentage 1. Far advanced with cavity 34 33.7 2. Fat advanced without cavity 12 11.9 3. Moderately advanced with cavity 19 18.8 4. Moderately advanced without cavity 31 30.7 5. Minimal active 5 4.9 The results of the microscopic examination of the different methods are summarized in Table 4. The direct smear microscopy yielded 64.3% (191/297) negative smears compared to the clorox-treated and sputufluol-treated sputum specimens with 54.2% (161/297) and 53.9% (160/297) negative smears respectively (p <0.001). Table 4. Results of microscopic examination in 297 sputum specimen prepared by different smear methods Microscopic Examination Results* Direct Smear Clorox treated Sputufluol treated Results No. % No. % No. % Negative 191 64.3 161 54.2 160 53.9 Rare 9 3.0 7 2.4 7 2.4 Few 28 9.4 16 5.4 19 6.4 Numerous 69 23.3 113 38.0 111 37.4 Total 297 100.0 297 100.0 297 100.0 p value ( 0.001) Positive results were noted in only 106 specimens of direct smears while more specimens yielded positive results when concentrated, 136 and 137 positive smears respectively for clorox and sputufluol. Most of these positive results were noted and recorded as numerous in the two concentrated methods than the direct smear, 37.38% compared to 23.3 % (p <0.001). When the results were lumped as either positive or negative alone, the percentage of positivity of the direct smear was 35.7% (106/297); that of clorox and sputufluol were 45.8% and 46.1% respectively, (p <0.001). Table 5 matched the results of the different methods utilized. The direct smear yielded more negative results compared to both concentration techniques. Concentration of the sputa increased the positivity rates of the direct method. An additional 33 more specimens which were negative with the direct smear were found positive with the clorox technique and 35 more with statistically significant results at p < 0.001. However, there were 3 clorox-treated specimens and 4 sputufluol-treated specimens that were negative but turned out positive with the direct smear method (Table 6). Table 5. Matched data between direct and concentration methods Clorox (Sputufluol)* Negative Positive Direct Smear Rare Few Numerous Total Negative 158 (156) 5 (4) 6 (7) 22 (24) 191 Positive Rare 1 (3) 1 (1) 4 (2) 3 (3) 9 Few 1 (1) 0 (2) 4 (5) 23 (20) 28 Numerous 1 (0) 2 (5) 65 (64) 69 Total 161 (160) 6 (7) 16 (19) 113(111) 297 *The numbers enclosed in the parenthesis refer to sputufluol Table 6. Correlation between direct and concentration methods Clorox (Sputufluol)* Negative Positive Total Negative 158 (156) 33 (35) 191 Positive 3 (4) 103 (102) 106 Total 161 (160) 136 (137) 297 *The numbers in parenthesis refer to sputufluol From the above results 2 positive results were observed more in specimens which were concentrated with either clorox or sputufluol than .the usual direct smear (Table 6). The kappa statistics for agreement beyond chance alone at 95% confidence interval between the direct smear and clorox was 0.59 and between direct and sputufluol, 0.57. There was a high degree of discordance between direct and both concentration methods/ Comparable results were obtained when clorox and sputufluol were matched as seen in Table 7. There was no difference between the total number of negative results (160 against 161) and the cumulative number of positive results (136 versus 137). At 95% confidence interval, the kappa statistics of agreement beyond chance alone between the two methods was 0.80, showing a high degree of concordance between the two concentration methods. Table 7. Matched data between clorox and sputufluol methods* Sputufluol Negative Positive Rare Few Numerous Total Negative 152 2 2 5 161 Rare 3 2 1 1 7 Few 2 2 9 3 16 Numerous 3 1 7 102 113 Total 160 7 18 111 297 *There was no statistical difference between these two methods (p value > 0.05) Culture was positive only in 16.8% (17/101) of all the study patients. The growth of Mycobacterium tuberculosis was observed only in the specimens subjected to sputufluol. There was no growth in those treated with clorox. Incidentally there were three patients with atypical mycobacteria noted in culture. The species were not identified. DISCUSSION A case of pulmonary tuberculosis is referred to by the World Health Organization as a patient with bacteriologically confirmed disease.17 For underdeveloped countries and in remote areas where culture facilities are unavailable, a bacteriologically confirmed disease is that in which a patient is found positive with acid-fast bacilli in the sputum by microscopy. The smear can establish a presumptive diagnosis of TB. The chances of finding the acid-fast bacilli in a smear increase with the concentration of the bacilli in the specimen. Several authors4,7,8 utilized different concentration techniques to increase the yield of positive acid-fast bacilli in the sputum and other body fluids, Table 8. It was generally recommended that addition of an alkali to the sputa can digest fibrous tissues, blood cells and other extraneous materials leaving a sediment, after centrifugation, consisting entirely of the tubercle bacilli. 7,8,9,15 Most of these studies were, however, time consuming and expensive. No statistical analysis was done in most of these studies to confirm their findings. This study utilized sodium hypochlorite, which is locally available as clorox and an imported one sputufluol and compared the results with the usual direct method. As a preliminary report this study showed that concentration methods were far superior to the direct smear in the recovery of acid-fast bacilli in the sputum. There were more positive results with both concentration methods than the direct smear method (p < 0.001). Conversely, the latter method yielded more negative results than the former two methods (p > 0.001). Table 8. Different concentration methods used in smears Authors Year Agents Used Petroff4 1915 4% sodium hydroxide Oliver and Reusser8 1942 Clorox (Sodium hypochlorite) Muller and Chermock6 1945 Phenol autoclave Krasnow and Wayne4 1946 Trisodium phosphate Krasnow et al5 1965 Trisodium phosphate with Zephiran Kubica and Kohn7 1963 N-acetyl-L-cysteine with sodium hydroxide A local study by Tech16 compared the direct method and the concentration method using sodium hydroxide. The results showed that the direct smear method screened more positive results than the sodium hydroxide. This s tudy however was uncontrolled with no statistical analysis done. In another major institution, a study in 197417 reported a 41.8% positivity rate among far-advanced cases of tuberculosis using the direct method. Foreign literature has cited different posit ivity rates from 30 to as high as 88% utilizing fluorescent microscopy. 15,18,19 Filho and Fonseca20 reported a 36.1% positivity rate using sodium hypochlorite with centrifugation and a 44.3% rate upon the addition of xylol as a flotation agent. Their results were comparable to this present study. This study was however uncontrolled. The degree of agreement between the direct and concentration methods was also computed. There was a lesser degree of concordance between the direct and both the concentration techniques. However, when both the concentration methods were compared, the degree of concordance between clorox and sputufluol was high at a kappa value of 0.80. From these preliminary results, one can use either of the two concentration methods in the laboratory. However, considering the cost and availability of the agents, clorox may substitute for the imported sputufluol. Clorox is a relatively cheap solution and is readily available in the rural areas. It is also noted that indeed this reagent is bactericidal considering the absence of growth in culture of all the specimens treated with it. This can be advantageous because in laboratories without biologic safety cabinets, this method can minimize infection among laboratory personnel. Thus, the material is safe for handling. In the Philippine General Hospital, there is ongoing use of this agent, clorox, but at a lower concentration (0.02%) in the recovery of the tubercle bacilli in culture. Another study will have to be undertaken this time to improve the yield in culture of the tubercle bacilli in this hospital as part of its thrust to function as a level III as well as level I mycobacteriology laboratory. The authors would like to suggest this method to the Department of Health as an alternative to the direct method in epidemiological case-finding activities and thereby complement the department's goal o t eradicate tuberculosis by the year 2000. 2 This must however be on the background of good technical expertise and adequate laboratory facilities in the peripheral health centers in the country. In this connection the authors, in cooperation with the Department of Health, will be conducting an anti-TB control project in an urban barangay in San Andres Bukid with the implementation of this method in case-finding activities in the said community and in monitoring the patient's response and progress to treatment. SUMMARY In summary, a total of 297 sputa were collected from 101 subjects .with clinical and radiological evidence of pulmonary tuberculosis. Mean age of these patients was.43.6 with ages ranging from 17-76 years o1d. The results showed that the positivity rate of the direct smear was 35.7%; that of clorox was 45.8% and 46.1% for sputufluol. Statistical significance was noted between the direct method and the two concentration methods as to the recovery of acid-fast bacilli in the sputum p < 0.001 There was also a great degree of discordance between these two methods (direct versus concentration). In addition, there was no statistical significance noted when the two concentration methods clorox against sputufluol were compared (p. > 0.05). Clorox can therefore be a. useful substitute to the more expensive sputufluol in this hospital to increase the yield of AFB in the sputum smear and should replace the direct method in epidemiological ease finding activities. Acknowledgement The authors would like to extend their sincere gratitude to the following people who helped in the completion of this project: Undersecretary Manuel Roxas, who supplied us with anti-TB medications, Mr. Eliseo Tura and Mrs. Medy Episcope our medical technologist and Miss Cynthia Cordero, the statistician of the project. REFERENCES 1. WHO/Japan International Tuberculosis Course: Tuberculosis Control. The Research Institute of Tuberculosis, JapanAntituberculosisAssociation, April 1979. 2. Department of Health, Manila, Philippines. National TuberculosisPrevalenceSurvey,1987 3. Tomas K. Tuberculosis case finding and chemotherapy, World Health Organization, 1979:7 4. Krasnow I, Wayne L. Sputum digestion: The mortality rate of tubercle bacilli in various digestion systems. The Am J ClinPathol 1966; 45(3):352-355. 5. Krasnow I, Kidd GC. The effect of a buffer wash of sputum sediments digested with zephiran trisodium phosphate on the recovery of acid-fast bacilli. Am J Clin Pathol 1965; 44:238-240. 6. Muller HE, Chermock RL. A rapid staining technique for acid-fast organisms. J Lab Clin Med 1945; 30:169-171. 7. Kubica GP, Kaufmann AJ, Dye WE. Comments on the use of the new mucolytic agents; N-Acetyl-L-Cysteine, as a s putum digestant for the isolation of mycobacteria. Am Rev Respir Dis 1964; 89:284-286, 8. Oliver J, Reusser TR. Rapid method for the concentration of tubercle bacilli. Am Rev Respir Dis 1942; 45:450-452. 9. Tarshis MS, Lewis WG. Use of clorox and tri-sodium phosphate in demonstration of acid-fast bacilli in sputum. Am J Clin Pathol 1949;19:688. 10. David HL. Bacteriology of mycobacteriosis. DHEW Public No. (CDC) 76-8316. Center for Disease Control, Atlanta, Georgia. 1976 11. Kubica GP, DavidHL. The mycobacterium. In: Gradwohls Clinical Laboratory Methods and Diagnosis. AC Sonnenwith, L Jarett (eds) 8th Edition. CV Mosby Co. 1980. 12. Wayne LG. Microbiology of tuberculosis. In: Pulmonary Diseases and Disorders AP.Fishman (ed) McGraw Hill Book Company, 1980. 13. Paul and Juhl's Essentials of Roentgen Interpretation. Harper and Row, 4 th edition, 1981. 14. American Thoracic Society. Diagnostic Standards and Classification of Tuberculosis and other Mycobactetial Diseases (14th edition), November, 1980. 15. Kubica GP, Gross WM, Hawkins JE, .Sommers HM, Vestal AL, Wayne LG. Laboratory services for mycobacterial diseases. Am Rev Respir Dis 1975; 112: 775. 16. Tech, DM. A comparative study of the smear methods in the identification of acid-fast bacilli from the sputum. JPMA 1965; 41(9):641-645. 17. Cruz BV, Morales CT, Manalo FM. Sputum microscopy in an emergency TB hospital: A study of the results in 2021 male cases in the Quezon Institute (1974-1976). Chest Dis 1976. 18. Boyd JC, Mart J. Decreasing reliability of acid-fast smear: Techniques for detection of tuberculosis . Ann Intern Med 197582:489-492. 19. Strumpf J, Tsang A, Sayre JW. Re-evaluation of sputum staining for the diagnosis of pulmonary tuberculo sis. Am Rev Respir Dis 1979;119: 599-602. 20. Filho CP, Fonseca KS. The flotation method for detection of tubercle bacilli in sputum smears. Tubercle 1979; 60:105-107.
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