Quick Links: Tuberculosis | Hansen’s Disease | Nontuberculous Mycobacteria | Recovery, Detection, and Identification of Mycobacteria at the DSHS Laboratory | Susceptibility Testing of Mycobacteria | 

^{A nine-banded armadillo (Dasypus novemcinctus) foraging in undergrowth in Belton, Texas. Armadillos likely acquired M. leprae from humans about 500 years ago. Photo by Lily Miller on Unsplash} 

The genus Mycobacteria includes many species, including the pathogenic bacilli known to cause serious diseases such as tuberculosis in mammals (M. tuberculosis complex) and Hansen’s Disease (M. leprae and M. lepromatosis) in humans. Mycobacteria may also be referred to as acid-fast bacilli (AFB), which refers to the cells’ resistance to decolorization during a staining procedure used to identify them. 
The M. tuberculosis complex (MTBC) refers to a group of genetically similar mycobacteria species that cause TB in both humans and animals: M. tuberculosis, M. bovis, M. bovis Bacillus Calmette–Guérin (BCG), and others. Other known pathogenic mycobacteria, referred to as Nontuberculous Mycobacteria (NTM) do not cause TB or Hansen’s disease, but can cause opportunistic diseases in people with existing lung diseases or compromised immune systems. 

Tuberculosis

Several species of Mycobacteria can cause TB. Mycobacterium tuberculosis, M. africanum, and M. canetti cause human TB. While M. tuberculosis is the most common cause of TB in humans, M. africanum and M. canetti are most commonly isolated from patients in Africa, or of African ancestry. M. bovis can infect humans, domestic and wild cattle and goats. M. microti is a rodent pathogen that is known to cause TB in immunocompromised human patients. M. caprae has been isolated only from goats, and M. pinnipedii infects seals. 

TB mycobacteria are spread from person to person through the air. The bacterium usually attacks the lungs but can also attack any part of the body such as the kidney, spine, and brain. Not everyone infected with TB mycobacteria becomes sick. As a result, two TB-related conditions exist: latent TB infection (LTBI), which is not contagious, and TB disease, which is. If not treated properly, TB disease can be fatal.
Increasing rates of antibiotic resistance in TB mycobacteria has complicated the management and treatment of the disease in humans and animals. 
Confirmed and suspected cases of tuberculosis are reportable in Texas.  
 

TB Testing at DSHS Austin Laboratory

The Laboratory carries out identification, genotyping, and drug susceptibility testing of M. tuberculosis complex. Please refer to the DSHS’ LTSM test menu for more details on M. tuberculosis testing at the Laboratory. General specimen submission guidance is available on DSHS’ specimen submission and shipping requirements pages.   
More information on TB and TB data and statistics in Texas may be found at:
DSHS - Frequently Asked Questions About TB (texas.gov)
Texas DSHS TB Program - TB Data and Statistics

Hansen’s Disease 

Mycobacterium leprae and M. lepromatosis cause Hansen’s disease (formerly known as leprosy). Infection by M. leprae or M. lepromatosis can lead to damage to the skin, eyes, and nerves. Peripheral nerve damage can result in an inability to feel pain, which if not treated may lead to loss of extremities due to repeated injuries or infection. 
Hansen’s disease has a low pathogenicity and contagiousness: it is spread through prolonged close contact with an infected person. Most people (approximately 95 percent) who get infected by the mycobacteria do not develop the disease. It can take many years for disease symptoms to develop. The clinical features of Hansen’s disease can vary widely from person to person and geographically. Genetic evidence indicates that an individual’s immune responses play a role in how the disease progresses. 
Hansen’s disease is rare in the United States, with approximately 150 newly diagnosed and reported cases each year. It is endemic in some populations of nine-banded armadillos in the southern U.S., so is considered a zoonotic disease in these areas. Transmission to people can occur when an infected armadillo is handled or eaten. It is believed that armadillos acquired M. leprae from humans approximately 500 years ago.

Hansen’s Disease Testing at DSHS Austin Laboratory 

The DSHS Laboratory does not test for Hansen’s Disease. DSHS suggests that specimens be submitted to the National Hansen’s Disease Program in Baton Rouge, Louisiana.

For more information on Hansen’s disease visit: 
Texas DSHS Hansen's Disease Program
Hansen's Disease (Leprosy) | CDC
National Hansen's Disease (Leprosy) Program Caring and Curing Since 1894 | HRSA

Nontuberculous Mycobacteria 

Other known pathogenic mycobacteria, referred to as Nontuberculous Mycobacteria (NTM) include M. avium, M. kansasii, M. abscessus, M. fortuitum, M. marinum, M. chelonae, and M. haemophilum.
NTM do not cause tuberculosis or Hansen’s disease but can cause lung, skin, skeletal, and disseminated chronic diseases that resemble tuberculosis. 
Nontuberculous mycobacteria are widely distributed in the environment, being found in soil and fresh surface waters. They are opportunistic pathogens that cause illness in people with existing lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis, or who have compromised immune systems. 
Mycobacterium avium complex (MAC), accounts for more than 80 percent of all cases of NTM lung disease in the United States. Recent studies indicate the number of new NTM infections in the U.S. is increasing, particularly in older, immunocompromised populations    . 
NTM skin infections are associated with traumatic injuries and medical or cosmetic procedures that disrupt the skin’s physical barrier. M. abscessus and M. chelonae infections of skin and soft tissues are most commonly associated with cutting or abrading the skin such as occurs with tattooing, body piercing, implants, dermabrasion, liposuction, or pedicures. Mycobacterial infections transmitted through acupuncture needles have also been documented.

NTM Testing at DSHS Austin Laboratory 

Nontuberculosis mycobacteria identification and susceptibility testing is available at the DSHS Austin Laboratory. Please refer to the LTSM test menu, found here, for more details on referred Acid Fast Bacilli (AFB) isolate identification testing at the laboratory and AFB specimen submission requirements.
For more information on NTM Infections, visit:
Nontuberculous Mycobacteria (NTM) Infections | HAI | CDC

Recovery, Detection, and Identification of Mycobacteria at the DSHS Laboratory

Mycobacteria are notorious for their extremely slow rate of cell division, with the doubling time for M. tuberculosis being 24 hours and 14 days for M. leprae. Such slow growth rates of mycobacteria require their prolonged incubation and observation to detect presence and growth.

Microscopy/Direct Observation

Direct microscopic examination of acid-fast stained smears is the most rapid method for detecting acid fast bacilli (AFB) in clinical specimens. For maximum sensitivity, smears are examined using the Auramine-O fluorochrome stain (A.K.A. Truant’s stain). Even though a positive acid-fast smear indicates that AFB are present, culturing/recovering organisms from the clinical specimen is required since negative smear results are not diagnostic and positive smear results are not specific since a positive AFB smear may be due to acid-fast organisms that are not M. tuberculosis.

Culture/Recovery   

Recovery of AFB from clinical specimens by growth in broth or on solid media allows for their identification. The Laboratory uses a combination of liquid and solid media to optimize AFB recovery. Cultured growth is identified by high performance liquid chromatography (HPLC) and/or biochemical tests, and results are reported immediately. M. tuberculosis has a relatively slow growth rate compared to other bacteria, so culture media are examined for a total of six weeks before being reported as negative for recovery or growth. The Laboratory uses both egg-based (Lowenstein-Jensen) and non-egg-based (Middlebrook 7H11) media for the recovery and isolation of all AFB cultures.

The Laboratory uses the automated BACTEC MGIT 960 culture system for the detection of AFB growth in liquid culture. The BACTEC MGIT 960 system has been shown to be both a rapid and sensitive method for the recovery of M. tuberculosis and other mycobacteria from clinical specimens.  The main benefit of using liquid culture is that the presence of some AFB species can be detected up to two weeks (on average) prior to the appearance of visible colonies on solid media.
The primary isolation of a small number of M. tuberculosis strains and other Mycobacterium species with special growth requirements (e.g., M. haemophilum) may require growth on solid media for optimal recovery. 

High Performance Liquid Chromatography 

This test is the Laboratory’s primary mode of identification for all mycobacteria. Identification of mycobacteria by HPLC is species-specific and is based on the separation of mycolic acids contained in the mycobacterial cell wall. The range of speciation and the sensitivity of the test are greater than that of genetic probe (without amplification) and conventional biochemical testing. 

Detection by HPLC testing can be performed directly on a clinical specimen regardless of its source. Direct HPLC is not highly sensitive and will not be conclusive on specimens that have negative or weakly AFB positive smears. For culture confirmation, pure, viable cultures on solid or in liquid media should be submitted to the lab for testing. Refer to the DSHS’ LTSM test menu for more details on HPLC testing of mycobacteria at the lab. 

Conventional Biochemical Tests

Once the isolate has been placed in a subgroup based on rate of its growth and pigmentation, identification to the species or complex level begins. Key biochemical tests useful for identifying the suspected species are selected and the organism’s biochemical profile is elucidated. The profile is compared to the reaction patterns of known species. If an unknown organism matches any one pattern exactly, the identification may be considered definitive for that species.   

Direct Nucleic Acid Amplification Testing 

Raw sputum or concentrated sputum sediment (from induced or natural/expectorated sputum) of patients suspected of having tuberculosis is tested by qualitative, nested real-time PCR NAAT for the presence of Mycobacterium tuberculosis DNA. 
In specimens where M. tuberculosis complex (MTBC) is detected, the Xpert® MTB/RIF Assay also detects the rifampin-resistance associated mutations of the rpoB gene. The test is intended to help diagnose pulmonary tuberculosis when used alongside clinical and other laboratory findings. 

Susceptibility Testing of Mycobacteria

BACTEC Method 

Pyrazinamide susceptibility testing of M. tuberculosis is carried out using the BACTEC ^_TM MGIT ^_TM 960 PZA kit. The kit includes a tube containing a modified Middlebrook 7H9 Broth, which supports the growth and detection of mycobacteria. The BD BACTEC MGIT 960 PZA Medium tube contains a fluorescent compound embedded in silicone on the bottom of a 16 x 100 mm round-bottom tube. The fluorescent compound is sensitive to the presence of oxygen dissolved in the broth. The initial concentration of dissolved oxygen quenches the emission from the compound, and little fluorescence can be detected. Later, actively growing and respiring microorganisms consume the oxygen, which allows the compound to fluoresce. 

The BD BACTEC ^TM MGIT ^TM 960 PZA Kit is a 4 to 21-day qualitative test. The test is based on growth of the M. tuberculosis isolate in a drug-containing tube compared to a drug-free tube (Growth Control). The BD BACTEC MGIT instrument monitors tubes for increased fluorescence. Analysis of fluorescence in the drug-containing tube compared to the fluorescence of the Growth Control tube is used by the instrument to determine susceptibility results. The BD BACTEC ^TM MGIT ^TM instrument automatically interprets these results and reports test organism as susceptible or resistant.

Antimicrobial Susceptibility Testing: Agar Proportion Method

Antimicrobial susceptibility testing is performed by agar proportion susceptibility. The assay compares the number of colonies growing on the surface of the drug-containing agar, relative to the number of colonies growing on the drug-free agar (control). Antibiotics are incorporated into cooled Middlebrook 7H10 agar. The agar is then poured into quadrant petri plates. One quadrant in each plate is reserved for the control. The organism is reported as resistant to a particular drug if the number of colonies on the drug quadrant is equal to or greater than 1% of the number of colonies growing on the drug-free control.

The Laboratory’s panel of drugs for agar proportion susceptibility testing of M. tuberculosis contains isoniazid, rifampin, ethambutol, streptomycin, rifabutin, ethionamide, kanamycin, capreomycin, and ofloxacin. Rifampin susceptibility for M. kansasii is also performed by the Laboratory.

Drug susceptibility testing of M. avium complex and other NTMs is not available at the DSHS Laboratory. Isolates may be forwarded by the Laboratory to a reference laboratory for susceptibility testing.