Direct Tuberculosis Culture in Selective Broth Without Decontamination or Centrifugation

Laboratorios de Investigación y Desarrollo, Departamento de Microbiología, Facultad de Ciencias, Universidad Peruana Cayetano Heredia, Lima, Perú; Asociación Benefica Prisma, Lima, Perú; Dept International Health, Johns Hopkins Bloomberg School of Hygiene and Public Health, Baltimore, USA; Dept Medical Microbiology, St George's Hospital Medical School, London; Wellcome Centre for Clinical Tropical Medicine and Department of Infectious Diseases and Immunity, Imperial College London Hammersmith Hospital Campus, UK

^* To whom correspondence should be addressed. Email: caevans{at}jhsph.edu.

	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 is not often possible in resource-poor settings. We therefore assessed the performance of Mycobacterium tuberculosis culture in unprocessed samples plated directly using tuberculosis-selective media compared to conventional culture using centrifuge-decontamination. Quadruplicate aliquots of H37RV strain were cultured in 7H9 broth with and without selective antimicrobials and after centrifuge-decontamination. Subsequently the 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 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 conventional culture (Kappa 0.84, P<0.001). Direct-culture is a simple, low-tech and rapid technique for diagnosing tuberculosis and determining drug susceptibility. Compared with conventional culture, direct-culture has reduced sensitivity because of bacterial overgrowth but in basic laboratories this may be outweighed by ease of use.