Study of Contaminants Growing on Lowenstein Jensen Media during Mycobacterium tuberculosis Culture from a Respiratory Speciality Hospital in Northern India

Abstract

Introduction: Lowenstein Jensen (LJ) culture contamination is one of the most frequent problems encountered during Mycobacterium tuberculosis (M. tuberculosis) culture. Contaminated cultures are repeated at an additional cost and thus hinder diagnosis. This problem is of more significant concern in Extrapulmonary (EP) samples which have very fewer bacilli loads. Unfortunately, our current contaminant knowledge from past studies is minimal, which is entirely based on pulmonary samples, and not on EP samples. Development of newer culture methods will remain incomplete unless we have a good knowledge about contaminant profiles from both types of samples. Aim: To isolate and identify bacterial and fungal contaminants growing on LJ media during M. tuberculosis complex culture in both pulmonary and EP samples. Materials and Methods: LJ media pairs (5074) were inoculated, of which 2030 were inoculated with pulmonary samples and 3044 with EP samples. Mycobacterial, non-Mycobacterial and fungal growth were differentiated based on characteristics like colony morphology (on Chocolate agar, blood agar, Mackonkey Agar and Sabouraud’s Dextrose Agar), staining (Gram stain and Ziehl Neelsen) and biochemical reactions (Indole, Urea, Citrate and Triple Sugar Iron). Statistical analysis was done using SPSS version 20 (IBM®, New York, NY, USA) and Chi-square test was performed. Results: Overall Contamination Rate (CR) was 2.2%. Individually, CR was 2.9% (60/2030) in pulmonary samples and 1.7% (52/3044) in EP samples (p<0.05). Of the total 303 organisms isolated in the study, 87.8% (266/303) were of bacterial origin and remaining 12.2% (37/303) were fungal. Bacteria isolated belonged to 12 different genera amongst which aerobic spore bearers (Bacillus spp) were the most common. All the 37 fungal isolates were moulds, of which 22 were Aspergillus spp (A. flavus, A. fumigatus and A. niger). Bacterial contaminants were more in pulmonary samples, whereas fungal in EP samples (p<0.05). All the contaminants were breakthrough as none grew as mixture along with acid-fast organisms. Conclusion: Breakthrough nature of contaminants indicates that they probably act by completely inhibiting the growth of any acid-fast bacilli present in the sample. This observation becomes quite relevant in EP samples wherein M. tuberculosis bacilli load is already very less. This M. tuberculosis growth masking can thus decrease the overall sensitivity of LJ culture.

Keywords

• bacteria
• culture media
• extrapulmonary samples
• pulmonary samples
• sputum