14.3 Acid-fast staining reagents

Direct microscopic examination to identify mycobacteria in a specimen after proper staining is common and useful in mycobacteriology. This is particularly important in resource-poor countries where culture may be difficult to carry out. Acid-fast staining may use carbol fuchsin-based methods, such as Kinyoun and Ziehl-Neelsen, and fluorochrome-based methods, such as auramine alone or auramine and rhodamine (Truant stain). These reagents can be prepared in-house; a number of commercial sources are also available (but not limited to), such as:

1. AlphaTec Systems. Common TB staining reagents are available along with TB processing reagents. Positive and negative staining control slides are also available.
   
2. BD Diagnostic Systems. These are Difco/BBL brand TB staining reagents. They are available in individual reagents and in kits. The kits include TB Stain Kit K (carbol fuchsin KF, decolorizer, and brilliant green), TB Stain Kit ZN (carbol fuchsin ZN, decolorizer, and methylene blue), TB Fluorescent Stain Kit M (auramine M, decolorizer TM, and potassium permanganate), TB Fluorescent Stain Kit T (auramine-rhodamine T, decolorizer TM, and potassium permanganate).
   
3. Remel Microbiology Products. All individual TB staining reagents are available. Two kits are TB Auramine O Stain Kit and TB Kinyoun AFB Stain Kit.
   
4. Aerospray TB Stainer (Wescor Biomedical Systems, Logan, UT, www.wescor.com; also PREVI Color TB in association with bioMérieux). This is an automated nondip-type stainer. The primary stain can be carbol fuchsin or fluorochrome. Excess primary stain is then spun off and the slide is rinsed with water to avoid precipitate formation. Decolorizer and counterstain are then applied stepwise. Finally, the slide is air dried. This automated stainer provides speed, economy, consistent performance, and no cross-contamination. Figure 14.1 shows an Aerospray TB Stainer.

Other methods, reagents, and instruments may also be available (such as the RAL Stainer, RAL Diagnostics, Montesquieu, Martillac, France; and QS-AFB, GG&B Co., Wichita Falls, TX). The user should investigate current commercially available products in his/her locale and consult company web sites and current literature.

14.4 Direct detection of mycobacteria from clinical specimens

14.5 Blood-culture recovery of mycobacteria

Regular blood cultures may also recover mycobacteria in the blood circulation [16], particularly rapidly growing mycobacteria (RGM), which can be cultivated after an incubation period of 3–5 days, similar to other fastidious organisms [24]. A Gram stain shows beaded Gram-positive or variable rods, and a positive acid-fast stain should be confirmative. The Isolator 10 system (Wampole Laboratories, Princeton, NJ) is particularly useful for recovering RGM because the intracellular mycobacteria are released from the lysed leukocytes and concentrated in the centrifuged sediment, and the culture plates support better growth of mycobacteria. Colony counts are also available from the plates. Other commercially available automated continuously monitoring culture systems (CMCS), such as BACTEC 9240, BacT/Alert 3D, and VersaTREK, can be used to recover rapid growers.

Blood-culture recovery of slowly growing mycobacteria commonly takes an incubation period of 2–3 weeks using the CMCS with appropriate media [11,26,39]. Some strains, however, may take up to 4–6 weeks. Mycobacterium avium and M. tuberculosis are the two most commonly recovered mycobacteria from patients with AIDS/HIV infection [2,28]. Mycobacterium avium bacteremia in AIDS/HIV used to be common in the United States [28], but is considerably less common now with better anti-HIV treatment. Mycobacterium tuberculosis bacteremia in AIDS/HIV is common in developing countries and carries a high and quick mortality [2,3,23,39].

14.6 Mycobacteria-culturing methods and systems

Traditional culture methods for mycobacteria mainly use Lowenstein–Jensen agar slants or bottles and Middlebrook 7H9, 7H10, and 7H11 agar plates or liquid. Many manufacturers supply them commercially. These culture media are simple, cheap, low technology, and easy to set up. They are particularly useful in resource-poor settings. The disadvantages are that they are labor-intensive and relatively less sensitive to recover M. tuberculosis. Many studies have consistently demonstrated that Lowenstein–Jensen agar has a sensitivity of 70–80%. Therefore, three specimens on different days are recommended to be cultured to reach a positive probability near 99%, the reciprocal probability of the product of three negative probabilities [1 − (1 − 0.75) × (1 − 0.75 × (1 − 0.75)].

14.6.1 BD Septi-Chek AFB System

The manual BD Septi-Chek AFB System (www.bd.com) system has been available since the early 1990s; it consists of a bottle with 20 mL culture media (7H9 broth) under an atmosphere of CO₂. The interior has a paddle containing three different media within a plastic tube, including Lowenstein–Jensen, Middlebrook 7H11, and chocolate agars. In general, nonacid-fast bacterial contaminants will grow on the chocolate agar whereas the mycobacteria grow on the remaining agars. The Septi-Chek AFB System performs reasonably well in comparison to other manual and automated systems (please check the availability of this product in your region or country, since, according to the manufacturer, this product line has been discontinued).

In the past two decades, the culture methods for mycobacteria have evolved to combine the traditional tube methods and newer liquid medium automated system in the United States. Several liquid automated systems are available; they are briefly described first, followed by comparative studies on their clinical performance.

14.6.2 BACTEC 460TB System

The BACTEC 460TB System (BD Microbiology Systems, www.bd.com) is the first automated system for mycobacteria culturing and testing. The system uses radiometric technology to sensitively detect the growth of mycobacteria in media. The system can be used with either 12B medium supplemented with PANTA (an antimicrobial agent solution) or 13A medium for whole blood. Due to the use of radioactive isotopes and the issue of its disposal, and the advent of comparable nonadiometric systems, this system is infrequently used now in most healthcare settings.

14.6.3 BACTEC MGIT 960 System

The Mycobacterial Growth Indicator Tube (MGIT) 960 (Figure 14.2; BD Microbiology Systems, www.bd.com) is a fully automated system for high-volume mycobacteria growth, detection, and susceptibility testing. It offers a capacity of 960 tubes for culture, safe operation, no radioactivity, and fast results. The system allows processing up to 8000 specimens per year, which meets the need of a medium to large hospital in the United States. The detection sensor uses fluorometric technology to detect oxygen consumption by the proliferating mycobacteria every 60 min to signal the positives as they occur. The EpiCenter Data Management System (an optional component) performs statistical analyses and accommodates different customer preferences. It also provides simple specimen tracking along with a flexible and dynamic reporting system for more in-depth data management needs. Depending on the hospital size, a smaller capacity MGIT 320 System is also available.

14.6.4 BacT/Alert 3D System

The BacT/Alert 3D System (bioMérieux, Durham, NC, www.biomerieux-usa.com

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