Virulence Mechanisms and Virulence Factors

Mycobacterium tuberculosis does not possess the classic bacterial virulence factors such as toxins, capsules and fimbriae. However, a number of structural and physiological properties of the bacterium are beginning to be recognized for their contribution to bacterial virulence and the pathology of tuberculosis.

MTB has special mechanisms for cell entry. The tubercle bacillus can bind directly to mannose receptors on macrophages via the cell wall-associated mannosylated glycolipid, LAM, or indirectly via certain complement receptors or Fc receptors.

MTB can grow intracellularly. This is an effective means of evading the immune system. In particular, antibodies and complement are ineffective. Once MTB is phagocytosed, it can inhibit phagosome-lysosome fusion. The exact mechanism used by MTB to accomplish this is not known but it is thought to be the result of a protein secreted by bacterium that modifies the phagosome membrane. The bacterium may remain in the phagosome or escape from the phagosome, in either case finding a protected environment for growth in the macrophage.

MTB interferes with the toxic effects of reactive oxygen intermediates produced in the process of phagocytosis by two mechanisms:

1. Compounds including glycolipids, sulfatides and LAM down regulate the oxidative cytotoxic mechanism.

2. Macrophage uptake via complement receptors may bypass the activation of a respiratory burst.

Antigen 85 complex. This complex is composed of a group of proteins secreted by MTB that are known to bind fibronectin. These proteins may aid in walling off the bacteria from the immune system and may facilitate tubercle formation although evidence of this is lacking.

Slow generation time. Because of MTB's slow generation time, the immune system may not readily recognize the bacteria or may not be triggered sufficiently to eliminate them. Many other chronic disease are caused by bacteria with slow generation times, for example,  slow-growing M. leprae causes leprosy, Treponema pallidum causes syphilis, and Borrelia burgdorferi causes Lyme disease.

High lipid concentration in cell wall, as mentioned previously, accounts for impermeability and resistance to antimicrobial agents, resistance to killing by acidic and alkaline compounds in both the intracellular and extracellular environment, and resistance to osmotic lysis via complement deposition and attack by lysozyme.

Cord factor. The cord factor is primarily associated with virulent strains of MTB. It is known to be toxic to mammalian cells and to be an inhibitor of PMN migration. However, its exact role in MTB virulence is unclear.
 

Clinical Identification and Diagnosis of Tuberculosis

The diagnosis of tuberculosis requires detection of acid-fast bacilli in sputum via the Ziehl-Neelsen method as previously described.

The organisms must then be cultured from sputum. First, the sputum sample is treated with NaOH. This kills other contaminating bacteria but does not kill the MTB present because cells are resistant to alkaline compounds by virtue of their lipid layer.

The media used for growth of MTB and the the resulting colony morphology have been described previously. However, methods of culturing can take 4-6 weeks to yield visible colonies. As a result, another method is commonly used call the BACTEC System. The media used in the BACTEC system contains radio-labeled palmitate as the sole carbon source. As MTB multiplies, it breaks down the palmitate and liberates radio-labeled CO₂. Using the BACTEC system, MTB growth can be detected in 9-16 days vs 4-6 weeks using conventional media.

Skin Testing is performed as the tuberculin or Mantoux test.  PPD (purified protein derivative) is employed as the test antigen in the Mantoux test. PPD is generated by boiling a culture of MTB, specifically Old Tuberculin (OT). 5 TU (tuberculin units), which equals 0.000lmg of PPD, in a 0.1 ml volume is intracutaneously injected in the forearm. The test is read within 48-72 hours.

Administering the Mantoux test. CDC.
 

The test is considered positive if the diameter of the resulting lesion is 10 mm or greater. The lesion is characterized by erythema (redness) and swelling and induration (raised and hard). 90% of people that have a lesion of 10 mm or greater are currently infected with MTB or have been previously exposed to MTB. 100% of people that have a lesion of 15 mm or greater are currently infected with MTB or have been previously exposed to MTB.

False positive tests usually manifest themselves as lesser reactions. These lesser reactions could indicate prior exposure or infection with other mycobacteria or vaccination with BCG. However, in places were the vaccine is not used, lesser reactions should be regarded as highly suspicious.

False negatives are more rare than false positives but are especially common in AIDS patients as they have an impaired CMI response. Other conditions such as malnutrition, steroids, etc., can rarely result in a false negative reaction.
 

Tuberculosis Treatment

Because administration of a single drug often leads to the development of a bacterial population resistant to that drug, effective regimens for the treatment of TB must contain multiple drugs to which the organisms are susceptible. When two or more drugs are used simultaneously, each helps prevent the emergence of tubercle bacilli resistant to the others. However, when the in vitro susceptibility of a patient's isolate is not known, which is generally the case at the beginning of therapy, selecting two agents to which the patient's isolate is likely to be susceptible can be difficult, and improper selection of drugs may subsequently result in the development of additional drug-resistant organisms.

Hence, tuberculosis is usually treated with  four different antimicrobial agents The course of drug therapy  usually lasts from 6-9 months. The most commonly used drugs are rifampin (RIF) isoniazid (INH), pyrazinamide (PZA ) and ethambutol (EMB) or streptomycin (SM). When adherence with the regimen is assured, this four-drug regimen is highly effective. Based on the prevalence and characteristics of drug-resistant organisms, at least 95% of patients will receive an adequate regimen (at least two drugs to which their organisms are susceptible) if this four-drug regimen is used at the beginning of therapy. Furthermore, a patient who is treated with the four-drug regimen, but who defaults therapy, is more likely to be cured and not relapse when compared with a patient treated for the same length of time with a three-drug regimen.
 

Drugs used to treat TB disease. From left to right isoniazid, rifampin, pyrazinamide, and ethambutol. Streptomycin (not shown) is given by injection. CDC.

Prevention

A vaccine against MTB is available. It is called BCG (Bacillus of Calmette and Guerin, named after the two Frenchmen that developed it). BCG consists of a live attenuated strain derived from Mycobacterium bovis. This strain of Mycobacterium has remained avirulent for over 60 years.

The vaccine is not 100% effective. Studies suggest a 60-80% effective rate in children.

The vaccine is not administered in the U.S. for several reasons:
� The vaccine cannot circumvent disease reactivation in previously exposed individuals.
� The vaccine does not prevent infection, only disease. Therefore, the entire population would have to be vaccinated if the vaccine was to be considered efficacious.
� Vaccination may complicate the way the tuberculin skin test is read in this country. In places that do not vaccinate, the skin test may be used to monitor the effectiveness of antibiotic therapy.

Multidrug-Resistant Tuberculosis (MDR TB) and Extensively Drug-Resistant Tuberculosis  (XDR TB)

Resistance to anti-TB drugs can occur when these drugs are misused or mismanaged. Examples include when patients do not complete their full course of treatment; when health-care providers prescribe the wrong treatment, the wrong dose, or length of time for taking the drugs; when the supply of drugs is not always available; or when the drugs are of poor quality.

Multidrug-resistant tuberculosis (MDR TB) is TB that is resistant to at least two of the best anti-TB drugs, isoniazid and rifampicin. These drugs are considered first-line drugs and are used to treat all persons with TB disease.

Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB. XDR TB is defined as TB which is resistant to isoniazid and rifampin, plus resistant to any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin). Because XDR TB is resistant to first-line and second-line drugs, patients are left with less effective treatment options, and cases often have worse treatment outcomes.

Both MDR TB and XDR TB are more common in TB patients that do not take their medicines regularly or as prescribed, or who experience reactivation of TB disease after having taken TB medicine in the past.

Persons with HIV infection or other conditions that can compromise the immune system are at highest risk for MDR TB and XDR TB. They are more likely to develop TB disease once infected and have a higher risk of death from disease.

The risk of acquiring XDR TB in the United States appears to be relatively low. However, it is important to acknowledge the ease at which TB can spread. But long as XDR TB exists, the U.S. is at some risk.

The risk of acquiring MDR TB in the United States is < 0.7% in U.S.-born persons but higher in foreign-born persons. Since 1998, the percentage of U.S.-born patients with MDR TB has remained fairly constant. However, of the total number of reported primary MDR TB cases, the proportion occurring in foreign-born persons increased from 25% (103 of 407) in 1993 to 80% (73 of 91) in 2006.

The Centers for Disease Control (CDC) Division of Tuberculosis Elimination advises how to prevent or minimize the development of MDR strains of M. tuberculosis.

� The most important thing a patient can do is to take all of their medications exactly as prescribed by a health care provider. No doses should be missed and treatment should not be stopped early. Patients should tell their health care provider if they are having trouble taking the medications. If patients plan to travel, they should talk to their health care providers and make sure they have enough medicine to last while away.

� Health care providers can help prevent MDR TB by quickly diagnosing cases, following recommended treatment guidelines, monitoring patients� response to treatment, and making sure therapy is completed.