Comparison Of Sputum Pretreatment For Isolation Of Mycobacterium Tuberculosis

Abstract

Recovery of mycobacteria from sputum and other contaminated clinical specimens require their treatment with special chemical agents to remove the commensal flora and to break down the mucus (Reider, et al., 1998). A variety of decontamination agents and procedures have been developed. The most widely used is the alkaline sodium hydroxide method originally proposed by Petroff and which carries his name (Reider, et al., 2007). The present study was designed to evaluate the methods that used for the stopping of the action of the 4% NaOH by neutralization after its application for the desired time. The aims of this work were to compare recovery of mycobacteria from sputum, to determine the susceptibility to the first line anti-tuberculous drugs of Mycobacterium tuberculosisisolated from new TB patients and to identify the isolates to the species level using phenotypic methods. A total of one hundred specimens were collected during the period from January to august 2006. One specimen from each patient was treated with sodium hydroxide NaOH 4% for 20 minutes and divided into three aliquots the first one was washed with phosphate buffer saline PBS pH 6.8 the second was neutralized with hydrochloric acid HCl 1N and the third one was inoculated directly on Kudoh acidic media. Recovery rates of mycobacteria from targeted subjects were found to be 92% when neutralized with 1 N HCl, 95% whenwashed with PBS pH 6.8 and 89% when directly inoculated on Kudoh acidified medium. With contamination rate 4%, 6.3% and 5.5% respectively. 77% of isolates were identified as Mycobacterium tuberculosis complex, 2% identified as mycobacteria other than tuberculosis and the rest 21% were unidentified. MDR within TB new cases was found to be 5.2%. The study concludes that every method have an excellent recovery rate with quite acceptable contamination rates and can be incorporated in the routinely used techniques in the national tuberculosis reference laboratory with especial attention for the internal quality control procedures. The study recommended that the specimens which contain blood are better to be neutralized with PBS because it is not important to determine the reaction end point. Smears that made from NaOH sediments werewashed off during staining, therefore no need for their preparation.