Role of Bleach Concentration Method for Detection of Acid-fast Bacilli (AFB) in Sputum using Conventional Ziehl-Neelsen (ZN) Staining Technique

Sputum microscopy is the most important tool for the detection of Mycobacterium tuberculosis in peripheral laboratories. Sodium hypochlorite concentration technique prior to sputum microscopy may improve the detection of AFB bacilli over direct Ziehl Neelsen’s staining method. A prospective study was conducted at Kasturba Medical College, Manipal. Consecutive sputum samples were collected for 3 months. ZN stained smears were made directly of fresh specimens and of specimens that were processed with 3.5% sodium hypochlorite (NaOCl), from each patient. Then, ZN stained smears were observed under light microscopy. A total of 239 samples were collected. The yield of positive smears with ZN staining after NaOCl centrifugation was higher than that with microscopy after direct ZN stain. The percentage increase yield of ZN staining after NaOCl centrifugation over direct ZN staining was 3.3% (8/239). Bleach centrifugation can help in increasing the yield of positivity in sputum smear.


INTRODUCTION
With millions of people being affected annually, tuberculosis (TB) is reckoned as a global health crisis for both developing and developed countries. 1Tuberculosis is one of the oldest diseases known to mankind and a significant cause of mortality and morbidity around the world is caused by Mycobacterium tuberculosis complex. 2 In developing countries such as India, sputum microscopy with Ziehl-Neelsen (ZN) staining is used to identify tuberculosis because it is simple, inexpensive and provides rapid results. 3,4leach (sodium hypochlorite, NaOCl) can be used as an ideal chemical processing agent in a resource-poor country.To enhance the yield of AFB for diagnosing pulmonary tuberculosis, digestion of sputum sample with bleach is suggested by some researchers. 5,6This agent is commonly available, affordable, and its disinfectant properties could enhance infection control in laboratories lacking proper biosafety equipment. 7

MATERIALS AND METHODS
This study was conducted from February 2019 to April 2019 in the Department of Microbiology, Kasturba Medical College, Manipal, Karnataka.
Patients presented with productive cough lasting more than 2 weeks duration were included in this study.The pre-analytical processing of sputum sample and sample anonymization had been done.Smear was made from each specimen for direct smear microscopy and was stained using the hot ZN method.The remainder of the specimen was transferred to a 15-mL disposable plastic conical tube with an equal volume of 3.5% commercial bleach (Emplura) added to the tube and was vortexed for 2-3 sec.The tube was then allowed to stand for 10-15 minutes at room temperature and then shaken again intermittently every 5 minutes.An equal quantity of distilled water was then added to the tube.The same was centrifuged at 3000 rpm for 15 minutes.After discarding the supernatant, the pellet was suspended in a few drops of the remaining fluid.By using a sterile glass pipette, 1-2 drops were transferred to a glass slide.Using the ZN stain for light microscopy, a smear was made and stained.

DISCUSSION
In India, TB remains to be a major public health problem.It is one of the leading causes of morbidity and mortality worldwide. 2n developing countries, sputum microscopy is the fastest, cheapest and the most widely used screening method for detection of TB in peripheral laboratories. 8Application of 3.5% bleach can inactivate the bacteria without altering their structure so that even if the bacteria get killed, they can be stained and observed under microscope.This provides a greater safety for laboratory personnel. 3leach has an oxidative effect, which disrupts the enzymatic processes of Mycobacteria, hence act as an effective disinfectant. 9It enhances biosafety by reducing aerosolization. 10Additionally, bleach centrifugation is known to increase smear sensitivity due to multiple factors.After the treatment with bleach, there are attributable changes in the surface properties (like, charge and hydrophobicity) of the mycobacteria.The process of denaturation of sputum constituents also leads to flocculation with subsequent increase in the sedimentation rate of mycobacteria. 11he standard method for concentration is the NALC-NaOH (N-acetyl-L-cysteine-sodium hydroxide) method which is technically demanding for a remote laboratory, and costs about 1 USD (INR 80 approx) per clinical specimen. 12NaOCl is however available at prices almost negligible (INR 1 / sample) as compared to NALC-NaOH method.At such cheap prices this easy concentration technique may be used for rapid and safe screening of tuberculosis in the remote areas.It does not require any in house reagent preparation and storage.But, this method is not suitable if culture is contemplated since hypochlorite kills acid fast bacilli.
In our study, no such hindrance to detection was noted.All positive cases by direct ZN staining were also detected by bleach centrifugation.Bleach centrifugation detected additional positives which were missed by direct ZN staining.Increase in positivity was found to be most for mucoid samples.For purulent, mucopurulent and blood-tinged samples, it did not make any significant difference.This finding is however in contrary to that of Bonnet M et al., who found that bleach centrifugation technique produced significant increase in positivity for purulent samples only among HIV patients. 13In the study conducted by Angeby KA et al.NaOCl concentration method yielded significantly more number of positive cases when compared with direct smear microscopy but differences between mucoid, purulent and mucopurulent samples was not evaluated. 14Bruchfeld J et al. reported similar results to Angeby KA et al. but on HIV patients. 15ur disagreement with Bonnet M et al. could be influenced by the difference in patient profile (HIV vs non-specific) but such a finding requires further corroboration from other studies. 13s noted by Saxena S et al., we also found that bleach sedimentation technique provided a clearer background for better observation.This, along with concentration by centrifugation may have contributed to improved grading after bleach centrifugation technique. 16leach centrifugation technique increases acid fast bacilli detection in sputum samples but as per this study it does so significantly in mucoid samples only.Bleach sedimentation provides clearer background for visualization and improves smear grading.Being a much cheaper and easier alternative, it may be implemented for high volume AFB screening at remote areas, though its effectiveness beyond mucoid samples may be disputed.

Table 1 .
Increased positivity by ZN staining in different types of sputum sample after bleach centrifugation (n = 239)

Table 2 .
Comparison between ZN staining of mucoid samples with and without hypochlorite (n = 89)

Table 3 .
Comparison between ZN staining of mucopurulent samples with and without hypochlorite (n = 45)

Table 4 .
Comparison between ZN staining of blood-tinged samples with and without hypochlorite (n = 13) Results of McNemar test: Chi-squared statistic = 0.000, P value = 1.000