Novel method for clearing red blood cell debris from BacT/ALERT^® blood culture medium for improved microscopic and antimycobacterial drug susceptibility test results

Abstract

Even though automation in mycobacterial culture has immensely improved the detection of organisms, identification of species and antimycobacterial susceptibility testing from blood culture bottles remain cumbersome and error-prone due to the presence of intact red blood cells (RBCs). The removal or lysis of these RBCs and excessive protein from the blood components could theoretically help improve this process. The present study reports an effective method that uses ammonium chloride (NH(4)Cl) and Triton X-100 to lyse the RBCs in blood culture medium. The method was optimized by preparing various concentrations of NH(4)Cl and Triton X-100, and incubation conditions, leading to eight protocols. The lysis protocol with a concentration of 150 mM of NH(4)Cl, 0.5% Triton X-100, and 1% potassium bicarbonate, pH 7.0, and incubation at 37 degrees C for 15 min was found to be optimal. This method not only made the culture medium clear, the protein concentration decreased from 753.5+/-39.4 to 53.2+/-4.2 mg/mL in the M. tuberculosis-spiked culture medium and in the blood culture medium inoculated with the blood from tuberculosis patients. The method had no adverse effect on mycobacteria, and no depletion of M. tuberculosis colony-forming units was found. The lysate could be used for antimycobacterial susceptibility testing with no difficulty in setting the mycobacterial concentration of inoculum to 0.5 McFarland standards. Furthermore, this method had the added advantage in the microscopy and molecular methods for the speciation of Mycobacterium sp.

In-vitro antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay

“…In the terminal stages of AIDS, where CD 4 + lymphocyte counts falls below 100, not only M. tuberculosis but several NTM also cause infection and can be isolated from blood, tissue, sputum and fecal samples [ 19 ]. The NTM disease not necessarily remains localized, but cause disseminated infection such as M. avium-intracellulare complex (MAC) [ 20 ]. The prognosis for the MAC infected AIDS patient is usually poor, largely due to the inherent resistance of these organisms to most of the available anti-tubercular drugs and have high toxicity.…”

Loop-mediated isothermal amplification assay for rapid and sensitive diagnosis of tuberculosis

“…maintained e 2 M. terrae (2) Lab. maintained e 2 M. kansasii (2) Lab. maintained e 2 M. chelonae (2) Lab.…”

“…Though smear microscopy is the simplest and most rapid diagnostic procedure currently available but its sensitivity is very low and requires at least 5 Â 10 3 bacilli/ml in the clinical samples which is a very solemn issue specially in extra-pulmonary (EPTB) samples. 2 The automated liquid culture based Mycobacteria Growth Indicator Tube (MGIT) system has reduced this time, but still it is not optimal. 3 In last 6e7 years, the diagnosis of TB has undergone a major breakthrough with the introduction of nucleic acid amplification (NAA) techniques.…”