Viruses that are a common cause of respiratory tract infection include adenoviruses, rhinoviruses, influenza, parainfluenza, enteroviruses, respiratory syncytial virus, retroviruses, herpes simplex, and cytomegalovirus. Because viruses need host DNA to replicate, they will not grow on artificial media. Therefore, viral cultures are inoculated onto cell cultures. These may be cancer cells grown in monolayers in the laboratory or cells taken from animal tissues and used to prepare a monolayer. Cell types commonly used for viral isolation are human diploid fibroblasts (HDF), HEp2 cells (epithelial cancer cells from the larynx), A549 cells (human lung cancer cells); primary monkey kidney cells (PMK), and rabbit kidney cells (RK). Cell cultures are inoculated and allowed to grow for one to three days at 36°C in 5-10% carbon dioxide. Within one to three days a characteristic cytopathic effect (CPE) can be seen by observing the cells under a microscope.
Mycobacterium tuberculosis is a respiratory infection commonly transmitted via the air to the lungs, where it thrives, causing fever, cough, and blood spitting. Pneumonia can also be caused by M. bovis, M. avium complex, and M. kansasii.
Most mycobacteria that cause pneumonia such as Mycobacterium tuberculosis grow very slowly requiring from two to six weeks for culture. Mycobacterium tuberculosis stains very poorly with Gram stain; therefore, acid-fast (light microscopy) and fluorescent staining methods are used to identify mycobacteria by direct microscopic examination of sputum. A smear can provide a presumptive diagnosis of mycobacterial disease; confirm that cultures growing on media are acid-fast; and demonstrate a patient's response to antibiotic therapy from post-treatment sputum cultures.
There are three staining methods commonly employed to test for mycobacteria. Two acid-fast stains, Ziehl-Neelsen and Kinyoun use light microscopy; the third method uses auramine or a combination of auramine and rhodamine and requires a fluorescent microscope. The advantage of fluorescent staining resides in the ability to examine much larger areas of the smear in a shorter period of time. Fluorescent staining is more sensitive and detects approximately 18% more cases. Its disadvantage is that the stain also detects organisms that are non-viable. While a positive finding provides a basis for initiating antibiotic treatment, the sensitivity of the direct smear is highly variable. Therefore, when acid-fast culture is requested, all sputum samples (meeting the laboratory's criteria for sputum) are cultured, even when the direct smear exam shows no evidence of acid-fast bacteria.
Sputum for culture of mycobacteria must be decontaminated. This is commonly done by adding a mucolytic agent such as N-acetyl-L-cysteine and a clearing agent, sodium hydroxide, to an equal amount of the sputum. The suspension is mixed and allowed to stand for 15 minutes, then phosphate buffered saline is added and the sample is centrifuged. The supernatant is decanted and the remaining sample is resuspended with bovine albumin and used for culture.
An acid-fast culture can detect as few as 10 to 100 CFU/mL of sputum. Culture media may be enriched with egg, albumin, or a mixture of salts, vitamins, cofactors, fatty acids, glucose, and other nutrients. Media also contain malachite green or antibiotics to retard the growth of other bacteria and yeast. The sputum should be inoculated on at least one solid medium such as American Thoracic Society or Lowenstein-Jensen and one liquid medium such as Middlebrook 7H9. Cultures are set up at several different temperatures and examined daily for several weeks to characterize the rate of growth. Colonies are subcultured and transferred to appropriate media for biochemical identification. This process can take several more weeks and therefore, other identification methods are often performed concurrently. These include analysis of cell wall fatty acids by either gas or high-performance liquid chromatography (HPLC) and DNA probe testing. High-performance liquid chromatography can rapidly identify the species of mycobacteria from cultures, but according to the CDC, laboratories that use HPLC report that the method requires a highly experienced technologist and usually takes a long time (about six months) for initial incorporation into their laboratories. DNA probe testing can be done on as little as a single colony and demonstrates a far more rapid turnaround time than biochemical testing. Probes are available for many species but not all mycobacterium, and may be falsely negative (i.e., a low hybridization rate) if contaminating organisms are present. For these reasons, this method is used in conjunction with biochemical testing.
Other microorganisms that cause various types of lower respiratory tract infections also require special culture or staining procedures to grow and identify. For example, Pneumocystis carinii in bronchial lavage, sputum, or lung biopsy samples is detected by observing the organisms with special stains such as methenamine silver stain or a fluorescent monoclonal antibody stain. Pneumocystis carnii causes pneumonia in people with weakened immune systems, such as people with AIDS, and does not grow in culture. The diagnosis is based on the results of these stains, the patient's symptoms, and medical history.
Victoria E. DeMoranville, The Gale Group Inc., Gale, Detroit,