Tuberculosis diagnosis and multidrug resistance testing by direct sputum culture in selective broth without decontamination or centrifugation

Abstract

Tuberculosis culture usually requires sputum decontamination and centrifugation to prevent cultures from being overgrown by contaminating bacteria and fungi. However, decontamination destroys many tuberculous bacilli, and centrifugation often is not possible in resource-poor settings. We therefore assessed the performance of Mycobacterium tuberculosis culture with unprocessed samples plated directly by using tuberculosis-selective media and compared this procedure to conventional culture using centrifuge decontamination. Quadruplicate aliquots of strain H37RV were cultured in 7H9 broth with and without selective antimicrobials and after centrifuge decontamination. The subsequent comparison was made with 715 sputum samples. Split paired sputum samples were cultured conventionally with centrifuge decontamination and by direct culture in tuberculosis-selective media containing antibiotics. Centrifuge decontamination reduced tuberculosis H37RV colonies by 78% (P < 0.001), whereas direct culture in tuberculosis-selective media had no inhibitory effect. Similarly, in sputum cultures that were not overgrown by contaminants, conventional culture yielded fewer tuberculosis colonies than direct culture (P < 0.001). However, the sensitivity of conventional culture was greater than that of direct culture, because samples were less affected by contamination. Thus, of the 340 sputum samples that were tuberculosis culture positive, conventional culture detected 97%, whereas direct culture detected 81% (P < 0.001). Conventional and direct cultures both took a median of 8.0 days to diagnose tuberculosis (P = 0.8). In those direct cultures that detected drug resistance or susceptibility, there was a 97% agreement with the results of conventional culture (Kappa agreement statistic, 0.84; P < 0.001). Direct culture is a simple, low-technology, and rapid technique for diagnosing tuberculosis and determining drug susceptibility. Compared to that of conventional culture, direct culture has reduced sensitivity because of bacterial overgrowth, but in basic laboratories this deficit may be outweighed by the ease of use.

FIG. 1.

Overview of methodology.

FIG. 2.

Growth of Mycobacterium tuberculosis in direct and decontaminated culture. The comparison was made for those cultures not affected by contamination. The overall difference between the two groups are shown (P < 0.001). The individual P values for the dilutions are P = 0.14 (1:1), P < 0.001 (1:20), P < 0.001 (1:400), and P < 0.001 (1:8,000). Error bars indicate 95% confidence intervals. The numbers of CFU in both culture techniques appear to increase with increasing dilution, but this is simply an artifact caused by false-negative-contaminated cultures that occur more frequently at higher sample concentrations.

FIG. 3.

Number of days from sample processing until the detection of tuberculosis growth is shown for the samples that were positive in both direct culture and decontaminated culture. Five of 715 samples were read after the usual 35 days.

FIG. 4.

(a) Results for each of the eight culture wells are shown for the 715 sputum samples. (b) Results of all culture dilutions combined.