14: Mycobacteria


Xiang Yang Han

Department of Laboratory Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

14.1 Introduction

The practice of clinical mycobacteriology has changed significantly over the past several decades. In the United States, with the steady declining number of patients with tuberculosis, the workload on mycobacteria in most hospitals is declining. Larger better-equipped reference laboratories perform more mycobacteriology work. On the other hand, nontuberculous mycobacteria are assuming more significance due to better recognition and more opportunistic infections in the increasing immunocompromised population. Worldwide, however, tuberculosis remains one of the most common causes of fatal infections, exacerbated by the human immunodeficiency virus pandemic and emergence of drug-resistant Mycobacterium tuberculosis strains. Public and private enterprises are joining hands to tackle the global tuberculosis problems.

Better commercial supplies of various reagents, culturing systems, and molecular detection and identification methods have substantially improved the quality and timeliness of mycobacterial detection and identification. This chapter, built on its first edition, will summarize our current practice and methods. The methods or systems are described first, and when applicable, clinical evaluation studies are summarized.

14.2 Specimen processing

Most clinical specimens are nonsterile and may contain nonacid-fast bacteria that would out-grow any Mycobacterium spp. soon upon culture. Specimen processing is essential to remove or kill those contaminants and to concentrate mycobacteria so that the culture can be kept for several weeks for best recovery and detection of mycobacteria. Treatment with N-acetyl-L-cysteine (NALC), a mucolytic agent, and with sodium hydroxide (NaOH), a lytic base, serves these purposes. Upon this chemical treatment, the specimen pellet is then resuspended in phosphate-buffered saline for culture inoculation. These reagents can be prepared readily, and many hospital laboratories do so. Commercially available reagents are convenient and also provide more consistent results within and between institutions. Included in these commercial manufacturers (but not limited to) are:

  1. AlphaTec Systems (Vancouver, WA, www.alphatecsystems.com). The reagents for digestion and decontamination include NAC-PAC, NAC-PAC Red, and NAC-PAC- EA3. The buffers include neutralization buffer (XPR-plus) to neutralize 3% NaOH used in heavy processing and pellet resuspension buffer (NPC-67).
  2. BD MycoPrep (BD Diagnostic Systems, Sparks, MD, www.bd.com). This specimen digestion/decontamination kit consists of ten 75 mL bottles of NALC-NaOH solution and five packages of phosphate buffer (pH 6.8).
  3. Remel Microbiology Products (Lenexa, KS, www.remel.com). The digestants include TB Base Digestant (sodium hydroxide/sodium citrate) and TB Digestant (4% sodium hydroxide).

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.
Photo displaying an Aerospray TB stainer.

Figure 14.1 Aerospray TB Stainer.

(Courtesy of ELITech Group, Inc.).

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

14.4.1 GeneXpert MTB/RIF Test

The Xpert MTB/RIF system (Cepheid, Sunnyvale, CA, www.cepheid.com) was developed several years ago. It combines detection of M. tuberculosis (MTB) and resistance to rifampin (RIF) in a single assay using hemi-nested real-time polymerase chain reactions (PCR) to amplify an MTB-specific sequence of the rpoB gene, which is probed with molecular beacons for mutations within the RIF-resistance determining region. The system integrates sample processing and PCR in a disposable plastic cartridge containing all reagents required for bacterial lysis, nucleic acid extraction, amplification, and amplicon detection. The only manual step is the addition of a bactericidal buffer to the sputum specimen before transferring to the cartridge. The MTB/RIF cartridge is then inserted into the GeneXpert device; the run completes within 2 h. The cartridge is a closed system to avoid amplicom contamination. The system requires minimal technical experience and training and suits developing countries where the tuberculosis burden is particularly heavy.

The World Health Organization has adopted the Xpert MTB/RIF for implementation in resource-poor countries in campaigns against tuberculosis since 2010 [65]. As of March 31, 2013, a total of 1123 GeneXpert instruments (comprising of 6181 modules) and 2.3 million testing cartridges had been procured in 83 countries [65]. Xpert MTB/RIF received FDA clearance recently.

14.4.2 AMPLIFIED MTD2 Test

The AMPLIFIED MTD2 (M. tuberculosis Direct) Test (Hologic Gen-Probe Inc., San Diego, CA, www.gen-probe.com) detects MTB rRNA directly and rapidly from acid-fast smear-positive and negative specimens. The MTD test is an isothermal transcription-mediated amplification (TMA) method based on specific mycobacterial rRNA targets using DNA intermediates. The RNA amplicons produced are identified by hybridization protection assay using an acridinium ester-labeled MTB complex-specific DNA probe. The test is specific for the MTB complex. It is FDA-approved. The MTD test is intended for use with specimens from patients that are showing signs and symptoms consistent with active pulmonary tuberculosis. The test is used in conjunction with routine acid-fast bacillus smear and culture. The test has a turn-around time of 2.5–3.5 h.

14.4.3 COBAS TaqMan MTB Test

The COBAS TaqMan MTB Test (Roche Diagnostic Corp, Indianapolis, IN, www.roche.com) uses real-time PCR nucleic acid amplification and hydrolysis probes for the detection of MTB complex DNA in liquefied, decontaminated and concentrated human respiratory specimens, including expectorated and induced sputum and bronchial alveolar lavages (BAL). The test utilizes the AMPLICOR Respiratory Specimen Preparation Kit for manual specimen preparation and the COBAS TaqMan 48 Analyzer for automated amplification and detection. Each run can test 48 samples. This test is currently not available in the United States at the time of this writing.

14.4.4 LCxMycobacterium tuberculosis assay

The LCx Mycobacterium tuberculosis assay (Abbott Diagnostics Division, Abbott Park, Ill., www.Abbott.com) uses the ligase chain reaction for the amplification of a segment of a gene in the MTB complex. The LCx assay was designed for use with processed respiratory specimens.

14.4.5 Clinical evaluation of direct tuberculosis tests

Many studies have been carried out to assess the performance of these direct test systems, particularly the Xpert MTB/RIF in recent years (Table 14.1). Large-scale multinational studies have shown that the Xpert MTB/RIF system provides sensitive and specific detection of tuberculosis and RIF resistance directly from untreated sputum in less than 2 h with minimal hands-on time. Overall, when compared with cultures (liquid and solid media), it performs best in smear-positive pulmonary TB (>90% sensitivity) and modest in smear-negative pulmonary TB and extrapulmonary TB (>70% sensitivity). It also shortens detection time to 1 day instead of weeks. In particular, it shortens initiation of treatment for RIF-resistant MTB from nearly 2 months to a single day, which will likely help to contain further dissemination of the resistant MTB strains.

Table 14.1 Clinical evaluation of GeneXpert for detection of M. tuberculosis (MTB) and rifampin (RIF) resistance or susceptibility in comparison with culture methods

Study location/type and year Number of culture samples and type* Xpert detection of MTB
Sensitivity Specificity RIF
Peru, Azerbaijan, South Africa, India (2010) [7] 561 smear (+) sputa 551 (98.2) 604 of 609 200 of 205 (97.6) RIF-resistant
171 smear (−) sputa 124 (72.5) 504 of 514 (98.1) RIF-susceptible
Peru, Azerbaijan, South Africa, India, Philippines, Uganda (2011) [8] 648 smear (+) sputa 637 (98.3) 2846 of 2876 236 of 250 (94.4) RIF-resistant
385 smear (−) sputa 296 (76.9) 796 of 810 (98.3) RIF-susceptible
Spain (2011) [38] 85, most pulmonary 64 (75) 40 of 40 6 of 6
Germany (2011) [27] 45 extrapulmonary 35 (77.8) 425 of 433 No data
Turkey (2011) [66] 27 smear (+) pulmonary 27 (100) 319 of 319 1 of 1 RIF-resistant
35 smear (−) pulmonary 24 (69) 88 of 88 RIF-susceptible
48 extrapulmonary 25 (52.1)
Meta-analysis of 18 studies (2012) [12] 10,224 suspected TB specimens 90.4% 98.4% 94.1% RIF-resistant, 97.0% RIF-susceptible

+, positive; −, negative.

* Extrapulmonary cases did not have smear data.

 Specificity in a study was for all culture-negative specimens.

 Percent in parentheses.

Selected large-scale evaluations of the MTD2, LCx, and AMPLICOR systems are listed in Table 14.2. These studies show that these rapid tests perform well overall, particularly for the smear-positive pulmonary specimens. One study recommends that, for smear-negative specimens, three consecutive specimens be tested to improve the test yield with AMPLICOR [17].

Table 14.2 Clinical evaluations of MTD2 and AMPLICOR tests in comparison to culture methods

Study location and year Number of specimens (isolates) Test Sensitivity (%) Specificity (%) Note
Italy (2000) [48] 486 (167), pulmonary and extrapulmonary MTD2 85.7 for pulmonary
82.9 for extrapulmonary
100 for both Both tests comparable, culture to confirm
AMPLICOR 94.2 for pulmonary
85 for extra-pulmonary
100 for both
Singapore (1999) [60] 230 (72), all pulmonary MTD2 98.6 99.4 Three tests comparable
AMPLICOR 96.1 100
LCx 100 99.3
Spain (1998) [19] 682 (163) MTD2 94.7 for pulmonary
86.8 for extrapulmonary
100 for both MTD2 useful
Denmark, Switzerland, Germany, UK (1996) [5] 7194 (654) AMPLICOR 91.4 for smear (+)
60.9 for smear (−)
96.1 Enabling rapid diagnosis of MTB
France (six centers) (1995) [11] 2073 (184) AMPLICOR 94.5 for smear (+)
74 for smear (−)
83 for extrapulmonary
98 Rapid and easy to perform
Germany (1998) [17] 1681 (65) AMPLICOR 96.4 for smear (+)
45.5 for smear (−)
99.7 Smear (+) samples preferred
Australia (1999) [32] 2347 (152) LCx 98.5 for smear (+),
41.5 for smear (−)
99.9 Smear (+) samples preferred
Italy (1999) [57] 697 (128) AMPLICOR 78 Not stated Comparable two tests
LCx 88

A novel rapid test, available in some markets, to detect the LAM antigen (lipoarabinomannan) in urine samples is the Alere Determine TB LAM Ag test (Alere, Waltham, MA). Please consult the vendor for additional information and performance characteristics.

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.

Photo displaying the Mycobacterial Growth Indicator Tube (MGIT) 960 system.

Figure 14.2 The Mycobacterial Growth Indicator Tube (MGIT) 960 System.

(Courtesy and © Becton, Dickinson and Company.)

14.6.4 BacT/Alert 3D System

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

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Dec 10, 2017 | Posted by in MICROBIOLOGY | Comments Off on 14: Mycobacteria
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