Acid-fast staining of sputum or other specimens is the usual initial test (see Figure1). Either the Kinyoun version of the acid-fast stain or the older Ziehl-Neelsen version can be used. The acid-fast stain has low sensitivity, as evidenced by the finding that approximately 50% of “smear-negative” samples are “culture positive.” For rapid screening purposes, auramine stain, which can be visualized by fluorescence microscopy, is used.
Fig1. Mycobacterium tuberculosis—acid-fast stain. Long red rods of M. tuberculosis are seen on a blue background. (Used with permission from Dr. George Kubica, Public Health Image Library, Centers for Disease Control and Prevention.)
In addition to performing an acid-fast stain, the specimen should be cultured. After digestion of the specimen by treatment with NaOH and concentration by centrifugation, the material is cultured on special media, such as Löwenstein Jensen agar or Middlebrook agar, for up to 8 weeks. It will not grow on a blood agar plate.
In liquid BACTEC medium, radioactive metabolites are present, and growth can be detected by the production of radioactive carbon dioxide in about 2 weeks. A liquid medium is preferred for isolation because the organism grows more rap idly and reliably than it does on agar. If growth in the culture occurs, the organism can be identified by biochemical tests. For example, M. tuberculosis produces niacin, whereas almost no other mycobacteria do. It also produces catalase.
Nucleic acid amplification tests (NAATs) can be used to detect the presence of M. tuberculosis directly in clinical specimens such as sputum. NAATs are available that detect either the ribosomal RNA or the DNA of the organism. These tests are highly specific, but their sensitivity varies. In sputum specimens that are acid-fast stain positive, the sensitivity is high, but in “smear-negative” sputums, the sensitivity is significantly lower. These tests are quite useful in deciding whether to initiate therapy prior to obtaining the culture results.
Because drug resistance, especially to isoniazid (see later), is a problem, susceptibility tests should be performed. However, the organism grows very slowly, and susceptibility tests usually take several weeks, which is too long to guide the initial choice of drugs. To address this problem, molecular tests are available that detect mutations in the chromosomal genes that encode either the catalase gene, which mediates resistance to isoniazid, or the RNA polymerase gene, which mediates resistance to rifampin. Whole genome sequencing can detect genotypic changes that determine resistance to isoniazid, rifampin, ethambutol, and pyrazinamide.
A urine test for active tuberculosis is available. The test detects the lipoarabinomannan antigen with high sensitivity and specificity. Its main drawback is that it does not provide drug susceptibility, so additional testing is required.
The luciferase assay, which can detect drug-resistant organ isms in a few days, is also used. Luciferase is an enzyme isolated from fireflies that produces flashes of light in the presence of adenosine triphosphate (ATP). If the organism isolated from the patient is resistant, it will not be damaged by the drug (i.e., it will make a normal amount of ATP), and the luciferase will produce the normal amount of light. If the organism is sensitive to the drug, less ATP will be made and less light produced.
There are two approaches to the diagnosis of latent infections. One is the PPD skin test as described in the “Immunity & Hypersensitivity” section earlier in this chapter. Because there are problems both in the interpretation of the PPD test and with the person returning for the skin test to be read, a quantifiable laboratory-based test is valuable.
This laboratory test is an interferon-γ release assay (IGRA), and there are two versions available: QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB. In the IGRA assay, blood cells from the patient are exposed to antigens from M. tuberculosis, and the amount of interferon-γ released from the cells is measured. The sensitivity and specificity of the IGRA are as good as those of the PPD skin test. Because the antigens used in the test are specific for M. tuberculosis and are not present in BCG, the test is not influenced by whether a person has been previously immunized with the BCG vaccine.
Note that the IGRA and PPD tests are positive in both latent disease and in active tuberculosis, so any person with a positive test must be evaluated for the presence of active disease by obtaining a chest X-ray and a sputum sample.
