Detection and identification of mycobacteria by mycolic acid analysis of sputum specimens and young cultures

Abstract

Ziehl–Neelsen acid-fast staining and mycolic acid analysis of concentrated samples and Middlebrook 7H9 cultures were carried out on 127 sputum specimens to evaluate a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures and compare with mycobacterial detection using Lowenstein–Jensen (LJ) cultures. All samples were classified into five groups according to the number of acid-fast bacilli observed in the smear. The group of samples with 3+ acid-fast bacilli in the smear had the highest number of positive detections of mycolic acids in the concentrated samples and the Middlebrook 7H9 cultures (81.8 and 100%, respectively). The overall percentages of mycolic acid detection for both sample types were 43.2 and 91.3%, respectively. The mycolic acid analysis of the Middlebrook 7H9 cultures had the fewest false negative detections with respect to the LJ cultures. The analysis of fluorescent derivatives of mycolic acids, using HPLC, is useful for concentrated sputum samples with large number of bacilli (3+) and is preferred for Middlebrook 7H9 cultures, even for clinical specimens with a low number of bacilli. Furthermore, with this analytical method, the simultaneous detection and identification of mycobacteria is usually possible.

Introduction

The rapid detection and identification of mycobacteria is important for controlling and preventing tuberculosis. The diagnosis of tuberculosis may include a variety of laboratory methods, with acid-fast smear and mycobacterial culture being the most often used. Smear microscopy, in particular, is used by clinical laboratories due to its low-cost and rapid results. In our state (Nuevo Leon, Mexico), however, up to 11% of new cases of tuberculosis are reported with negative smear microscopy (personal communication, Departamento de Control Epidemiológico de los Servicios de Salud de Nuevo León, Mexico). Mycobacterial culture followed by biochemical testing is the most sensitive and specific method for detecting mycobacteria in clinical specimens. Pure strains, abundant growth, and long incubation times, however, are necessary. In many countries, the method of choice uses cultures in Lowenstein–Jensen (LJ) slants. High-performance liquid chromatography (HPLC) for mycobacterial lipids (mycolic acids) has also been described as an alternative method for identifying mycobacterial isolates from clinical specimens (Butler and Guthertz, 2001). The standardized method analyzes UV-absorbing p-bromophenacyl derivatives of mycolic acids (Butler and Guthertz, 2001, Butler et al., 1996), and mycolic acid pattern standards have been described for the HPLC identification of mycobacteria (Butler et al., 1999). Jost et al. (Jost et al., 1995) proposed the use of fluorescent 4-bromomethyl-6,7-dimethoxycoumarin derivatives of mycolic acids for differentiating mycobacterial species directly in smear-positive sputum specimens and in BACTEC 12B broth, thereby, increasing the sensitivity of detection 200-fold. Furthermore, our group showed the utility of the fluorescent derivatives of mycolic acids for drug susceptibility testing of Mycobacterium tuberculosis (Viader-Salvadó et al., 2001, Viader-Salvadó et al., 2000, Garza-González et al., 1997) and for detecting M. tuberculosis in clinical isolates and smear-positive concentrated sputum samples (Garza-González et al., 1998). This chromatographic method, used directly for clinical specimens, can identify the genus and species in less than 24 h after clinical specimens are received, while also detecting mycobacteria in samples with negative smear microscopy from patients with pauci-bacillary disease. Thus, we evaluated a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures.