Part III: Culture
introduction

Tuberculosis is a disease of global importance. One-third of the world population is estimated to have been infected with Mycobacterium tuberculosis and nine million new cases of tuberculosis arise each year. The tuberculosis crisis is likely to escalate since the human immunodeficiency (HIV) epidemic has triggered an even greater increase in the number of tuberculosis cases. The majority of tuberculosis patients are 15 to 45 years of age, persons in their most productive years of life. Tuberculosis kills over two million people world-wide each year, more than all other infectious diseases combined, including AIDS and malaria.

Transmission of tuberculosis is virtually entirely by droplet nucleii created through coughing by untreated persons suffering from pulmonary tuberculosis (the most common form) in a confined environment. Infected droplets remain airborne for a considerable time, and may be inhaled by susceptible persons.

Pulmonary tuberculosis usually occurs in the apex of the lungs. These develop cavities which contain large populations of tubercle bacilli that can be detected in a sputum specimen. Pulmonary tuberculosis is suggested by persistent productive cough for three weeks or longer, weight loss, night sweats and chest pain. The diagnosis can only be made reliably on demonstrating the presence of tubercle bacilli in the sputum by means of microscopy and/or culture in the laboratory.

The cornerstone of the laboratory diagnosis of tuberculosis is direct microscopic examination of appropriately stained sputum specimens for tubercle bacilli. Between 5 000 and 10 000 tubercle bacilli per millilitre of sputum are required for direct microscopy to be positive and only a proportion of tuberculosis patients harbour large enough numbers of organisms to be detected in this way. It is also virtually impossible to distinguish different mycobacterial species by microscopy. Patients who have positive smears carry the greatest number of tubercle bacilli, are the most infectious and are therefore the most important patients to detect early because they are responsible for spreading tuberculosis disease.

Sputum examination by microscopy is relatively quick, easy and inexpensive and must be performed on ll cases suspected of having tuberculosis. Smear microscopy is also used to monitor treatment progress and control programme outcome.

Examination by bacteriological culture provides the definitive diagnosis of tuberculosis. Depending on the decontamination method and the type of culture medium used, as few as ten viable tubercle bacilli can be detected. However, the usual microbiological techniques of plating clinical material on selective or differential culture media and sub-culturing to obtain pure cultures cannot be applied to tuberculosis bacteriology. Compared to other bacteria which typically reproduce within minutes, M. tuberculosis proliferate extremely slowly (generation time 18-24 hours). Furthermore, growth requirements are such that it will not grow on primary isolation on simple chemically defined media. The only media which allow abundant growth of M. tuberculosisare egg-enriched media containing glycerol and asparagine, and agar or liquid medium supplemented with serum or bovine albumin.

Culture increases the number of tuberculosis cases found, often by 30-50%, and detects cases earlier, often before they become infectious. Since culture techniques can detect few bacilli, the efficiency of diagnosing failures at the end of treatment can be improved considerably. Culture also provides the necessary material for drug susceptibility testing. Culture of specimens is, however, much more costly than microscopy and requires facilities for media preparation as well as skilled staff.

Culture should be used selectively, in the following order of priority: