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MYCOBACTERIUM GROUP
C. N. Peralta, RMT
KDCI 2021
Mycobacterium tuberculosis Complex
Mycobacterium tuberculosis
Mycobacterium bovis
MAJ OR G E NER A AND
SPE CI ES TO B E
CONSI DE R ED
Mycobacterium bovis BCG
Mycobacterium africanum
Mycobacterium caprae
Mycobacterium canettii
Mycobacterium microti
Mycobacterium pinnipedii
Nontuberculous Mycobacteria
Slow-Growing Nonphotochromogens
M. avium complex
M. avium
subsp. avium
subsp. silvaticum
subsp. paratuberculosis
M. intracellulare
MAJ OR G E NER A AND
SPE CI ES TO B E
CONSI DE R ED
M. celatum
M. ulcerans
M. gastri
M. genavense
M. haemophilum
M. malmoense
M. shimoidei
M. xenopi
M. heidelbergense
M. branderi
M. simiae
M. triplex
M. conspicuum
Photochromogens
• M. kansasii
• M. asiaticum
• M. marinum
• Scotochromogens
• M. szulgai
• M. scrofulaceum
MAJOR
GENERA AND
SPECIES TO BE
CONSIDERED
• M. interjectum
• M. gordonae
• M. cookii
• M. hiberniae
• M. lentiflavum
• M. conspicuum
• M. heckeshornense
• M. tusciae
• M. kubicae
• M. ulcerans
• M. bohemicum
MAJ OR G E NER A AND
SPE CI ES TO B E
CONSI DE R ED
Noncultivatable
M. leprae
Rapid-Growing, Potentially Pathogenic
MAJOR GENERA
AND SPECIES TO BE
CONSIDERED
•M. fortuitum
•M. chelonae
•M. abscessus subsp. abscessus
•M. abscessus subsp. bolletii
•M. smegmatis
•M. peregrinum
•M. immunogenum
•M. mucogenicum
•M. neworleansense
•M. brisbanense
•M. senegalense
•M. porcinum
•M. houstonense
•M. boenickei
•M. wolinskyi
•M. goodii
•M. septicum
•M. mageritense
•M. canariasense
•M. alvei
•M. novocastrense
•M. cosmeticum
•M. boenickei
•M. canariasense
•M. setense
MYCOBACTERIUM
• Mycobacterium are aerobic (although some may grow in reduced oxygen concentrations)
• non–spore forming (except for M. marinum)
• Nonmotile
• very thin
• slightly curved or straight rods (0.2 to 0.6 Å~ 1 to 10 μm)
• Some species may display a branching morphology
• Mycobacterium is the only genus in the Mycobacteriaceae family (Actinomycetales order,
Actinomycetes class)
• Genera that are closely related to Mycobacterium include Nocardia, Rhodococcus,
Tsukamurella and Gordonia.
MYCOBACTERIUM
• grow more slowly than most other human pathogenic bacteria
because of their hydrophobic cell surface
• organisms tend to clump so that nutrients are not easily allowed into the cell.
• single cell’s generation time (the time required for a cell to divide into two
independent cells) may range from approximately 20 hours to 36 hours for
Mycobacterium ulcerans.
• formation of visible colonies in 2 to 60 days at optimum temperature
MYCOBACTERIUM
• Mycobacterium spp. have an unusual cell wall structure.
• cell wall contains N-glycolylmuramic acid instead of N-acetylmuramic acid
• it has a very high lipid content, which creates a hydrophobic permeability barrier.
• mycobacteria are difficult to stain with commonly used basic aniline dyes, such as those used in Gram
staining.
• Although these organisms cannot be readily Gram stained, they generally are considered gram
positive
• they resist decolorization with acidified alcohol (3% hydrochloric acid) after prolonged application of a
basic fuchsin dye or with heating of this dye after its application
• This important property of mycobacteria, which derives from their cell wall structure, is referred to as
acid fastness; this characteristic distinguishes mycobacteria from other genera.
• Rapid-growing mycobacteria (RGMs) may partially or completely lose this characteristic as a result of
their growth characteristics.
MYCOBACTERIUM
• genus Mycobacterium includes more than 100 recognized or proposed species.
• produce a spectrum of infections in humans and animals ranging from localized lesions to
disseminated disease.
• Some species cause only human infections, and others have been isolated from a wide variety of
animals.
• Many species are also found in water and soil.
• mycobacteria can be divided into two major groups, based on fundamental differences in
epidemiology and association with disease:
• Mycobacterium tuberculosis complex
• those referred to as nontuberculous mycobacteria (NTM)
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• Tuberculosis was endemic in animals in the Paleolithic period, long before it
ever affected humans.
• This disease (also called consumption) has been known in all ages and
climates.
• tuberculosis was the subject of a hymn in a sacred text from India dating
from 2500 BC, and DNA unique to Mycobacterium tuberculosis was
identified in lesions from the lung in 1000-year-old human remains found in
Peru.
GENERAL CHARACTERISTICS
• In the clinical microbiology laboratory, the term complex frequently is
used to describe two or more species for which distinction is
complicated and has little or no medical importance.
• The mycobacterial species that occur in humans and belong to:
• M. tuberculosis complex
• M. tuberculosis
• M. bovis
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
• M. bovis BCG,
• M. africanum
• M. caprae
• M. microti
• M. canettii
• M. pinnipedii.
• All of these species are capable of causing tuberculosis.
• It should be noted that species identification might be required for
epidemiologic and public health reasons.
• The organisms that belong to the M. tuberculosis complex are
considered slow growers, and colonies are nonpigmented.
Epidemiology
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
• M. tuberculosis is the cause of most cases of human tuberculosis,
particularly in developed countries.
• An estimated 1.7 billion people, or one third of the world’s
population, are infected with M. tuberculosis.
• This reservoir of infected individuals results in 8 million new
cases of tuberculosis and 2.9 million deaths annually.
• Tuberculosis continues to be a public health problem
• An additional complicating factor in the management of
tuberculosis is the increasing incidence of co-infection with the
human immunodeficiency virus (HIV).
• HIVassociated tuberculosis remains a significant challenge to
world health, with an estimated 1.1 million individuals living
with HIV-associated tuberculosis.
• tuberculosis typically is found among the poor, homeless,
intravenous (IV) drug users, alcoholics, the elderly, or medically
underserved populations
• Although the organisms belonging to the M. tuberculosis
complex have numerous characteristics in common, including
extreme genetic homogeneity, they differ in certain
epidemiologic aspects
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• Pathogenesis
• Inhalation of a single viable organism has been shown to lead to infection, although close contact is usually necessary.
• Of those who become infected with M. tuberculosis, 15% to 20% develop disease.
• The disease usually occurs some years after the initial infection, when the patient’s immune system breaks down for
some reason other than the presence of tuberculosis bacilli in the lung.
• In a small percentage of infected hosts, the disease becomes systemic, affecting a variety of organs.
• After ingestion of milk from infected cows, Mycobacterium bovis may penetrate the gastrointestinal mucosa or invade
the lymphatic tissue of the oropharynx.
• An attenuated strain of M. bovis, bacillus Calmette-Guérin (BCG), has been used extensively in many parts of the world
to immunize susceptible individuals against tuberculosis.
• Because mycobacteria are the classic examples of intracellular pathogens and the body’s response to BCG hinges on
cell-mediated immunoreactivity, immunized individuals are expected to react more aggressively against all antigens that
elicit cell-mediated immunity.
•
In rare cases, an unfortunate individual’s immune system is so compromised that it cannot handle the BCG, and
systemic BCG infection may develop.
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
SPECTRUM OF DISEASE
• Tuberculosis may mimic other diseases, such as pneumonia, neoplasm, or fungal infections.
• Clinical manifestations in patients infected with M. tuberculosis complex may range from
asymptomatic to acutely symptomatic.
• Patients who are symptomatic can have systemic symptoms
• pulmonary signs and symptoms
• signs and symptoms related to other organ involvement (e.g., the kidneys)
• a combination of these features.
• Cases of pulmonary disease caused by M. tuberculosis complex organisms are clinically,
radiologically, and pathologically indistinguishable.
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• Primary tuberculosis (PTB) typically is considered a disease of the
respiratory tract.
• Common presenting symptoms
• low-grade fever
• night sweats
• Fatigue
• anorexia (loss of appetite)
• weight loss.
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• A patient who presents with pulmonary tuberculosis usually has
• productive cough, along with low-grade fever
• Chills
• myalgias (aches)
• Sweating
• however, these signs and symptoms are similar for influenza, acute bronchitis, and
pneumonia
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• Upon respiratory infection with M. tuberculosis complex organisms, the cellular immune system T
cells and macrophages migrate to the lungs, and the organisms are phagocytized by the
macrophages.
• However, these organisms are capable of intracellular multiplication in the macrophages.
• Often the host is unable to eliminate the organisms, and the result is a systemic hypersensitivity to
Mycobacterium antigens.
• Granulomas or a hard tubercle forms in the lung from the lymphocytes, macrophages, and cellular
pathology, including giant cell formation (cellular fusion displaying multiple nuclei).
• If the Mycobacterium antigen concentration is high, the hypersensitivity reaction may result in tissue
necrosis, caused by enzymes released from the macrophages.
• In this case no granuloma forms, and a solid or semisolid, caseous material is left at the primary
lesion site.
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• In some patients infected with primary active tuberculosis, the disease may spread via the lymph
system or hematogenously, leading to meningeal or military (disseminated) tuberculosis.
• This most often occurs in patients with depressed or ineffective cellular immunity.
• in a small percentage of patients, organs besides the lungs can become involved after infection with
M. tuberculosis complex organisms
• These organs include the following:
•
•
•
•
•
•
•
•
Genitourinary tract
Lymph nodes (cervical lymphadenitis)
Central nervous system (meningitis)
Bone and joint (arthritis and osteomyelitis)
Peritoneum
Pericardium
Larynx
Pleural lining (pleuritis)
• Disseminated tuberculosis may be diagnosed by a positive tuberculin skin test
Ahmad, Clinical and developmental
immunology, 2011
A Koul et al. Nature 469, 483-490 (2011) doi:10.1038/nature09657
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• Patients also may have latent disease (i.e., they have no apparent signs, symptoms, or
pathologic condition).
• A patient with latent tuberculosis is not infectious and does not have active disease,
although the organism is present in granulomas.
• Patients with latent tuberculosis may progress to active disease (also referred to as
reactivation of tuberculosis) at any time.
• Reactivation tuberculosis typically occurs after an incident in which cellular immunity is
suppressed or damaged as a result of a change in life style or other health condition.
• Individuals infected with HIV are particularly susceptible to developing active tuberculosis.
• These patients are likely to have rapidly progressive primary disease instead of a subclinical
infection.
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• Diagnosing tuberculosis is more difficult in people infected with HIV, because chest radiographs of the
pulmonary disease often lack specificity, and patients frequently are anergic (lack a biologic response) to
tuberculin skin testing, a primary means of identifying individuals infected with M. tuberculosis.
• The tuberculin skin test, or purified protein derivative (PPD) test, is based on the premise that after
infection with M. tuberculosis, an individual develops a delayed hypersensitivity cell-mediated immunity to certain
antigenic components of the organism.
• To determine whether a person has been infected with M. tuberculosis, a culture extract of M. tuberculosis (i.e.,
PPD of tuberculin) is injected intracutaneously.
• After 48 to 72 hours, an infected individual shows a delayed hypersensitivity reaction to the PPD, characterized
by erythema (redness) and, most important, induration (firmness as a result of influx of immune cells).
• The diameter of induration is measured and then interpreted as to whether the patient has been infected with
M. tuberculosis; different interpretative criteria are used for different patient populations (e.g.,
immunosuppressed individuals, such as those infected with HIV).
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
T-Spot TB test (Oxford, Immunotec, United Kingdom) offers next-day results and does not
require a follow-up visit with a physician.
• The assay measures T cells that have been activated by Mycobacterium tuberculosis antigens.
• Peripheral blood mononuclear cells are incubated with M. tuberculosis-specific antigens stimulating
any sensitized T cells in the patient sample.
• T cell cytokines released in the sample are measured using antibody to capture them and then
detected with a secondary antibody conjugated to alkaline phosphatase.
• This assay should be interpreted in correlation with the patient’s signs and symptoms.
MYCOBACTERIUM TUBERCULOSIS
COMPLEX
• The PPD test is not 100% sensitive or specific, and a positive reaction to the skin test does not necessarily signify the presence
of disease.
• Because of these issues, a new test approved by the U.S. Food and Drug Administration (FDA) has become available. It is an
enzyme-linked immunosorbent assay (ELISA) called QuantiFERON-TB Gold (Cellestis Limited, Carnegie,Victoria, Australia).
• The assay measures a component of the cell-mediated immune response to M. tuberculosis to diagnose latent tuberculosis
infection and tuberculosis disease.
• It is based on the quantification of interferongamma released from sensitized lymphocytes in heparinized whole blood that has
been incubated overnight with a mixture of synthetic peptides simulating two proteins in M. tuberculosis.
• The test assesses responses to multiple antigens; it can be performed in a single patient visit; and it is less subject to reader bias
and error.
• An important feature is that the results of the assay are unaffected by previous BCG vaccination.
• Guidelines published by the Centers for Disease Control and Prevention (CDC) recommend the use of this assay in all
circumstances in which the tuberculin skin test currently is used (e.g., contact investigations and evaluation of recent immigrants).
• The guidelines also provide specific cautions for interpreting negative results in individuals from selected populations.
Epidemiology of Organisms Belonging to M. tuberculosis
Complex That Cause Human Infections
• M. tuberculosis
• Habitat:
• Patients with cavitary disease are primary reservoir
• Primary Route of Transmission:
• Person to person by inhalation of droplet nuclei: droplet nuclei containing
the organism (infectious aerosols, 1 to 5 μm) are produced when people
with pulmonary tuberculosis cough, sneeze, speak, or sing; infectious
aerosols may also be produced by manipulation of lesions or processing
of clinical specimens in the laboratory.
• Droplets are so small that air currents keep them airborne for long
periods; once inhaled, they are small enough to reach the lungs’ alveoli*
• Distribution:
• Worldwide
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
M. bovis
• Habitat:
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
• Humans and a wide range of host animals,
such as cattle, nonhuman primates, goats,
cats, buffalo, badgers,
possums, dogs,
pigs, and deer
• Primary Route of Transmission:
• Ingestion of contaminated milk from
infected cows; airborne transmission.
• Distribution:
• Worlwide
M. africanum
• Habitat:
• Humans
• Primary Route of Infection:
• Inhalation of droplet nuclei
• Ditribution:
• East and West Tropical Africa; some
cases have been identified in the
United States
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
M. Caprae
• Habitat:
• Humans rarely; predominately
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
infects a wide range of
Animals
• Primary Route of Infection:
• Inhalation of droplet nuclei
• Distribution:
• Europe
M. microti
• Habitat:
• Humans rarely; small animals (e.g., voles
and other wild rodents)
• Primary Route of Infection:
• Inhalation of droplet nuclei
• Distribution:
• Europe; Great Britain, Netherlands
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
M. canettii
• Habitat:
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
• Natural reservoir has not been
clearly defined. Rarely infects
humans.
• Primary Route of Infection:
• Unclear
• Distribution:
• Africa
M. pinnipedii
• Habitat:
• Humans rarely; predominantly infects a wide
range of animals
• Primary Route of Infection:
• Unclear
• Distribution:
• Europe
M Y C O B AC T E R I U M
TUBERCULOSIS
COMPLEX
NONTUBERCULOUS MYCOBACTERIA
• NTM include all mycobacterial species that do not belong to M. tuberculosis complex
Other Names That Have Been Used to Designate the Nontuberculous Mycobacteria
• Anonymous
• Atypical
• Unclassified
• Unknown
• Tuberculoid
• Environmental
• Opportunistic
• Mycobacteria other than tubercle bacilli (MOTT)
• NTM are present everywhere in the environment
• sometimes colonize the skin and respiratory and
gastrointestinal tracts of healthy individuals
NONTUBERCULOUS
M Y C O B AC T E R I A
• some mechanisms of infection appear to be trauma,
inhalation of infectious aerosols, and ingestion; a few
diseases are nosocomial or are acquired as an
iatrogenic infection
• NTM are not usually transmitted from person to
person, nor does isolation of these organisms
necessarily mean they are associated with a disease
process
NONTUBERCULOUS MYCOBACTERIA
• 1959 Runyon classified NTM into four groups based on the
phenotypic characteristics of the various species, most notably
the growth rate and colonial pigmentation
• Runyon’s system first categorizes the slow-growing NTM
(Runyon groups I to III) and then the rapid-growers (Runyon
group IV)
Runyon
Group
Group Name
Description
Number
NONTUBERCULOUS
M Y C O B AC T E R I A
I
Photochromogens
NTM colonies that develop pigment on
exposure to light after being grown in
the dark and take longer than 7 days to
appear on solid media
II
Scotochromogens
NTM colonies that develop pigment in
the dark or light and take longer than 7
days to appear on solid media
III
Nonphotochromog NTM colonies that are Nonpigmented
ens
regardless of whether they are grown
in the dark or light and take longer than
7 days to appear on solid media
IV
Rapid growers
• Runyon’s Classification
NTM colonies that grow on solid media
and take fewer than 7 days to appear
NONTUBERCULOUS MYCOBACTERIA
SLOW-GROWING NONTUBERCULOUS MYCOBACTERIA
• Three groups based on the phenotypic characteristics of the species.
• Mycobacterium spp. synthesize carotenoids (a group of yellow to red pigments) in varying amounts and
thus can be categorized into three groups based on the production of these pigments:
• Photochromogens: are slow-growing NTM that produce colonies that require light to form
pigment
• Scotochromogens: are slow-growing NTM that produce pigmented colonies whether grown
in the dark or the light
• Nonphotochromogens.: NTM that produce unpigmented colonies whether grown in the
dark or the light
NONTUBERCULOUS MYCOBACTERIA
• Photochromogens
• The photochromogens are slow-growing NTM that produce colonies that require light to
form pigment.
CHARACTERISTICS OF NONTUBERCULOUS
MYCOBACTERIA—PHOTOCHROMOGENS
Organism
Epidemiology
Pathology
Type of Infection
M. kansasii
Infection more common in white males;
natural reservoir is tap water; aerosols are
involved in transmission
Potentially pathogenic
Chronic pulmonary disease;
extrapulmonary diseases, such
as cervical lymphadenitis and
cutaneous disease
M. asiaticum
Not commonly encountered (primarily
seen in Australia)
Potentially pathogenic
Pulmonary disease
M. marinum
Natural reservoirs are freshwater and
saltwater as a result of contamination from
infected fish and other marine life.
Transmission is by contact with
contaminated water and organism entry by
means of trauma or small breaks in the
skin; associated with aquatic activity usually
involving fish
Potentially pathogenic
Cutaneous disease; bacteremia
M. intermedium
Unknown
Potentially pathogenic
Pulmonary disease
M. novocastrense
Unknown
Potentially pathogenic
Cutaneous disease
NONTUBERCULOUS MYCOBACTERIA
Scotochromogens
• The scotochromogens are slow-growing NTM that produce pigmented colonies whether grown in
the dark or the light.
• The epidemiology of the potentially pathogenic scotochromogens has not been definitively
described.
• In contrast to potentially pathogenic nonphotochromogens, these agents are rarely recovered in the
clinical laboratory.
C H A R AC T E R I S T I C S O F N O N T U B E R C U L O U S M Y C O B AC T E R I A — S C OTO C H RO M O G E N S
Organism
Epidemiology/Habitat
Pathogenicity
Type of infection
M. szulgai
Water and soil
Potentially pathogenic
Pulmonary disease, predominantly in
middle-aged men; cervical adenitis; bursitis
M. scrofulaceum
Raw milk, soil, water, dairy
products
Potentially pathogenic
Cervical adenitis in children, bacteremia,
pulmonary disease, skin infections
M. interjectum
Unknown
Potentially pathogenic
Chronic lymphadenitis, pulmonary disease
M. heckeshornense
Unknown
Potentially pathogenic
Pulmonary disease (rare)
M. tusciae
Unknown – isolated from tap
water
Potentially pathogenic
Cervical lymphadenitis (rare)
M. kubicae
Unknown
Potentially pathogenic
Pulmonary disease
M. gordonae
Tap water, water, soil
Nonpathogenic
NA
M. cookie
Sphagnum moss, surface waters
in New Zealand
Nonpathogenic
NA
M. hiberniae
Sphagnum moss, soil in Ireland
Nonpathogenic
NA
NONTUBERCULOUS MYCOBACTERIA
Nonphotochromogens
• are slow-growing NTM that produce unpigmented colonies whether grown in the dark or the light.
• Of the organisms in this group, M. terrae complex (M. terrae, M. triviale, and M. nonchromogenicum)
and M. gastri are considered nonpathogenic for humans. The other nonphotochromogens are
consideredpotentially pathogenic, and many are frequently recovered in the clinical laboratory.
• The nonphotochromogens belonging to Mycobacterium avium complex are frequently isolated in
the clinical laboratory and are able to cause infection in the human host.
C H A R AC T E R I S T I C S O F T H E N O N T U B E R C U L O U S
M Y C O B AC T E R I A — N O N P H OTO C H RO M O G E N S A N D S P E C I E S
C O N S I D E R E D P OT E N T I A L PAT H O G E N S
Organism
Epidemiolgy
Type of infection
M. avium complex
Environmental sources, including natural waters, and soil
Patients without AIDS: Pulmonary infections in
patients with preexisting pulmonary disease;
cervical
lymphadenitis; and disseminated disease* in
immunocompromised patients who are HIV
negative
Patients with AIDS: Disseminated disease
M. xenopi
Water, especially hot water taps in hospitals; believed to be
transmitted in aerosols
Primarily pulmonary infections in adults; less
common,
extrapulmonary infections (bone, lymph nodes,
sinus
tract) and disseminated disease
M. ulcerans
Stagnant tropical waters; also harbored in an aquatic
insect’s salivary glands; infections occur in tropical or
temperate climates
Indolent cutaneous and subcutaneous
infections
(African Buruli ulcer or Australian Bairnsdale
ulcer)
CH AR ACT E R IST ICS OF T H E NONT UB E R CULOUS
MY COB ACT E R IA — NONP HOTOCH ROMOGENS A ND
SPE CI ES CONSI DE R ED P OT E NT I AL PATH OG E NS
Organism
Epidemiolgy
Type of infection
M. malmoense
Most cases from England, Wales, and
Sweden. Rarely isolated from patients
infected with HIV. Little is known about
epidemiology; to date, isolated only
from humans and captured armadillos
Chronic pulmonary infections, primarily in patients
with preexisting disease; cervical lymphadenitis
in children; less common, infections of the skin
or bursae
M. genovense
Isolated from pet birds and dogs. Mode
of acquisition
unknown
Disseminated disease in patients with AIDS (wasting
disease characterized by fever, weight loss,
hepatosplenomegaly, anemia)
M. haemophilum
Unknown
Disseminated disease; cutaneous infections in
immunosuppressed adults; mild and limited skin
infections in preadolescence or early adolescence;
cervical lymphadenitis in children
M. heidelbergense
Unknown
Lymphadenitis in children; also isolated from sputum,
urine, and gastric aspirate
NONTUBERCULOUS MYCOBACTERIA
Mycobacterium avium Complex (MAC).
• Largely because of the increasing populations of immunosuppressed patients, the incidence of
infection caused by M. avium complex spp., as well as these organisms’ clinical significance, has
changed significantly since they were first recognized as human pathogens in the 1950s.
• The introduction of highly active antiretroviral therapy (HAART) has dramatically reduced the
infections caused by these organisms in patients with acquired immunodeficiency syndrome (AIDS).
NONTUBERCULOUS MYCOBACTERIA
General Characteristics
• Taxonomically, M. avium complex comprises
•
M. avium
•
M. intracellulare
•
M. avium subsp. Avium
•
M. avium subsp. Paratuberculosis
•
M. avium subsp. silvaticum (wood pigeon bacillus)
•
M. vulneris
•
M. marseillense
•
M. bouchedurhonense
•
M. timonense.
• The name M. avium subsp. hominissuis has been proposed for another subspecies capable of infecting humans.
• Although M. avium and M. intracellulare are clearly different organisms, they so closely resemble each other that the distinction
cannot be made by routine laboratory determinations or on clinical grounds.
NONTUBERCULOUS MYCOBACTERIA
Epidemiology and Pathogenesis
• MAC is an important pathogen in both immunocompromised and immunocompetent
populations
• MAC is particularly noteworthy for its potentially pathogenic role in pulmonary infections
in patients with AIDS and also in patients who are not infected with HIV.
• The organisms are ubiquitous in the environment and have been isolated from natural
water, soil, dairy products, pigs, chickens, cats, and dogs.
• it is generally accepted that natural waters serve as the major reservoir for most human
infections
NONTUBERCULOUS MYCOBACTERIA
• Infections caused by MAC are acquired by inhalation or ingestion.
• The pathogenesis of MAC infections is not clearly understood.
• The organisms are commonly associated with respiratory disease clinically similar to tuberculosis in
adults, lymphadenitis in children, and disseminated infection in patients with HIV.
• However, these organisms and other environmental NTM have extraordinary starvation survival.
They can persist well over a year in tap water, and MAC tolerates temperature extremes.
• In addition, similar to legionellae, M. avium can infect and replicate in protozoa. Amoebae-grown M.
avium is more invasive toward human epithelial and macrophage cells. MAC cultures can have an
opaque, a translucent, or a transparent colony morphology. Studies suggest that transparent colonies
are more virulent because they are more drug resistant, are isolated more frequently from the
blood of patients with AIDS, and appear more virulent in macrophage and animal models.
NONTUBERCULOUS MYCOBACTERIA
• M. avium subsp. paratuberculosis is known to cause an inflammatory bowel disease (known as
Johne’s disease) in cattle, sheep, and goats.
• It also has been isolated from the bowel mucosa of patients with Crohn’s disease, a chronic
inflammatory bowel disease of humans.
• The organism is extremely fastidious, seems to require a growth factor (mycobactin, produced by
other species of mycobacteria, such as M. phlei, a saprophytic strain) and may take as long as 6 to 18
months for primary isolation.
• Whether these and other mycobacteria actually contribute to development of Crohn’s disease or
are simply colonizing an environmental niche in the bowel of these patients remains to be
elucidated.
NONTUBERCULOUS MYCOBACTERIA
Clinical Spectrum of Disease
Other Nonphotochromogens
• Several other mycobacterial species that are considered nonphotochromogens are potentially
pathogenic in humans.
• newer species of mycobacteria that are nonphotochromogens have been described
• M. celatum
• M. conspicuum
• These newer agents appear to be potentially pathogenic in humans.
RAPIDLY GROWING NONTUBERCULOUS
MYCOBACTERIA (RGM)
• Mycobacteria that produce colonies on solid media in 7 days or
earlier constitute the second major group of NTM.
• Currently, approximately 70 species have been classified into this
group.
RAPIDLY GROWING NONTUBERCULOUS
MYCOBACTERIA (RGM)
General Characteristics
• The large group of organisms that constitute the RGM is divided into six
major groups of potentially pathogenic species, based on pigmentation and
molecular studies.
• Unlike the majority of other mycobacteria, most rapid-growers can grow
on routine bacteriologic media and on media specific for cultivation of
mycobacteria.
• On Gram staining, these organisms appear as weakly gram-positive rods
resembling diphtheroids.
RAPIDLY GROWING NONTUBERCULOUS
MYCOBACTERIA (RGM)
Epidemiology and Pathogenesis
• The rapidly growing mycobacteria considered potentially pathogenic can cause disease in
either healthy or immunocompromised patients.
• Like many other NTM, these organisms are ubiquitous in the environment and are present
worldwide.
• have been found in soil, marshes, rivers, and municipal water supplies (tap water) and in marine
and terrestrial life forms
• Infections caused by rapidly growing mycobacteria can be acquired in the community from
environmental sources
• can be nosocomial infections, resulting from medical interventions (including bone marrow
transplantation), wound infections, and catheter sepsis
RAPIDLY GROWING NONTUBERCULOUS
MYCOBACTERIA (RGM)
• on artificial media; these organisms are capable of infecting macrophages
• The smooth colonial phenotype typically is identified in biofilms and lacks infectivity
NONCULTIVATABLE NONTUBERCULOUS
MYCOBACTERIUM LEPRAE
• The nontuberculous mycobacterium M. leprae is a close relative of M.
tuberculosis.
• This organism causes leprosy (also called Hansen’s disease).
• Leprosy is a chronic disease of the skin, mucous membranes, and nerve
tissue.
• Leprosy remains a worldwide public health concern as a result of the development of
drug-resistant isolates.
NONCULTIVATABLE NONTUBERCULOUS
General Characteristics
• M. leprae has not yet been cultivated in vitro, although it can be cultivated in the armadillo and in
the footpads of mice.
• Molecular biologic techniques have provided most of the information about this organism’s genomic
structure and its various genes and their products.
• Although polymerase chain reaction (PCR) assays have been used to detect and identify M. leprae in
infected tissues, the technique thus far has not proved as effective diagnostically as anticipated in
indeterminate or paucibacillary (few organisms present) disease
• diagnosis of leprosy is based on distinct clinical manifestations, such as hypopigmented skin
lesions and peripheral nerve involvement, in conjunction with a skin smear that tests
positive for acid-fast bacilli
NONCULTIVATABLE NONTUBERCULOUS
Epidemiology and Pathogenesis
• Understanding of the epidemiology and pathogenesis of leprosy is hampered by the
inability to grow the organism in culture. In tropical countries, where the disease is most
prevalent, it may be acquired from infected humans; however, infectivity is very low.
• Prolonged close contact and the host’s immunologic status play roles in infectivity.
Epidemiology
• The primary reservoir for M. leprae is infected humans.
• The disease is transmitted person to person through inhalation or contact with infected
skin.
• The more important mode of transmission appears to be inhalation of M. leprae
discharged in the nasal secretions of an infected individual.
NONCULTIVATABLE NONTUBERCULOUS
Pathogenesis
• Although the host’s immune response to M. leprae plays a key role in control of infection, the
immune response is also responsible for the damage to skin and nerves; in other words, leprosy is
both a bacterial and an immunologic disease.
• After acquisition of M. leprae, the infection passes through many stages, which are characterized by
their histopathologic and clinical features.
• Although the infection has many intermediate stages, the two primary phases are a silent phase,
during which the leprosy bacilli multiply in the skin in macrophages, and an intermediate phase, in
which the bacilli multiply in peripheral nerves and begin to cause sensory impairment.
• More severe disease states may follow.
• A patient may recover spontaneously at any stage.
NONCULTIVATABLE NONTUBERCULOUS
• Spectrum of Disease
• Based on the host’s response, the spectrum of disease caused by M. leprae ranges from subclinical
infection to intermediate stages of disease to full-blown and serious clinical manifestations involving the
skin, upper respiratory system, testes, and peripheral nerves.
• The two major forms of the disease are a localized form, called tuberculoid leprosy, and a more
disseminated form, called lepromatous leprosy.
• Patients with lepromatous leprosy are anergic to M. leprae because of a defect in their cell-mediated
immunity.
• Because the organisms’ growth is unimpeded, these individuals develop extensive skin lesions containing
numerous acid-fast bacilli; the organisms can spill over into the blood and disseminate.
• In contrast, individuals with tuberculoid leprosy do not have an immune defect, so the disease is localized
to the skin and nerves; few organisms are observed in skin lesions.
• Most of the serious sequelae associated with leprosy are the result of this organism’s tropism for
peripheral nerves.
TUBERCULOID VS . LEPROMATOUS LEPROSY
CLINIC AL MANIFESTATIONS AND IMMUNOGENICITY
Lepromatous vs.Tuberculoid Leprosy
LEPROMATOUS LEPROSY (EARLY/LATE
STAGES)
LEPROMATOUS LEPROSY PRE- AND
POST-TREATMENT
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
• Specimens received by the laboratory for mycobacterial smear and culture must be handled in a safe
manner.
• Tuberculosis ranks high among laboratory-acquired infections
• laboratory and hospital administratorsbmust provide laboratory personnel with facilities, equipment, and
supplies that reduce this risk to a minimum.
• M. tuberculosis has a very low infective dose for humans (i.e., an infection rate of approximately 50%
with exposure to fewer than 10 acid-fast bacilli).
• All tuberculin-negative personnel should have a skin test at least annually.
• The CDC recommends Biosafety Level 2 practices, containment equipment, and facilities for
preparing acid-fast smears and culture for nonaerosolizing manipulations.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
• If M. tuberculosis is grown and then propagated and
manipulated, biologic safety cabinet (BSC) class II safety
precautions are required; however, Biosafety Level 3 practices
are recommended.
• BSC Level 3 practices are recommended for opening
centrifuge vials, adding reagents to biochemical testing medias,
and sonication; these practices include restricted laboratory
access, negative pressure airflow, and special personal protective
equipment (e.g., certified respirators).
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
SPECIMEN COLLECTION AND TRANSPORT
•
Acid-fast bacilli can infect almost any tissue or organ of the body.
•
Successful isolation of these organisms depends on the quality of the specimen obtained and the use of appropriate processing and culture
techniques by the mycobacteriology laboratory.
•
In suspected mycobacterial disease, as in all other infectious diseases, the diagnostic procedure begins at the patient’s bedside.
•
Collection of proper clinical specimens requires careful attention to detail by health care professionals.
•
Most specimens are respiratory samples
•
•
sputum, tracheal or bronchial aspirates, and specimens obtained by bronchial alveolar lavage
Other samples
•
urine, gastric aspirates, tissue (biopsy) specimens, cerebrospinal fluid (CSF), and pleural and pericardial fluid.
•
Blood or fecal specimens may be collected from immunocompromised patients.
•
Specimens should be collected in sterile, leakproof, disposable, and appropriately labeled containers without fixatives and placed in bags to
contain leakage.
•
If transport and processing will be delayed longer than 1 hour, all specimens except blood should be refrigerated at 4° C until processed.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
Pulmonary Specimens
• Pulmonary secretions may be obtained by any of the following methods:
•
spontaneously produced or induced sputum
•
gastric lavage
•
transtracheal aspiration
•
Bronchoscopy
•
laryngeal swabbing.
• Most specimens submitted for examination are sputum, aerosol-induced sputum, bronchoscopic aspirations, or gastric lavage
samples.
• Spontaneously produced sputum is the specimen of choice.
• To raise sputum, patients must be instructed to take a deep breath, hold it momentarily, and then cough deeply and vigorously.
• Patients must also be instructed to cover the mouth carefully while coughing and to discard tissues in an appropriate receptacle.
• Saliva and nasal secretions should not be collected, nor should the patient use oral antiseptics during the collection period.
• Sputum specimens must be free of food particles, residues, and other extraneous matter.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
• The aerosol (saline) induction procedure can best be done on ambulatory patients who are able to
follow instructions.
• Aerosol-induced sputum specimens have been collected from children as young as 5 years of age.
• This procedure should be performed in an enclosed area with appropriate airflow.
• Operators should wear particulate respirators and take appropriate safety measures to prevent
exposure.
• The patient is told that the procedure is being performed to induce coughing to raise sputum that
the patient cannot raise spontaneously and that the salt solution is irritating.
• The patient is instructed to inhale slowly and deeply through the mouth and to cough at will,
vigorously and deeply, coughing and expectorating into a collection tube.
• The procedure is discontinued if the patient fails to raise sputum after 10 minutes or feels any
discomfort.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
• Ten milliliters of sputum should be collected; if the patient continues to raise sputum, a second
specimen should be collected and submitted.
• Specimens should be delivered promptly to the laboratory and refrigerated if processing is delayed.
• Sputum collection guidelines recommend collection of an early morning specimen for 3 consecutive
days.
• In many cases the third specimen demonstrates minimal recovery of organisms, and this collection
may not be recommended in some laboratories.
• Pooled specimens are unacceptable because of an increased risk of contamination.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
Gastric Lavage Specimens
• Gastric lavage is used to collect sputum from patients who may have swallowed sputum during the
night.
• The procedure is limited to senile, nonambulatory patients; children younger than 3 years of age
(specimen of choice); and patients who fail to produce sputum by aerosol induction.
• The most desirable gastric lavage is collected at the patient’s bedside before the patient arises and
before exertion empties the stomach.
• Gastric lavage cannot be performed as an office or clinic procedure.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
• The collector should wear a cap, gown, and particulate respirator mask and should stand beside (not in front of) the patient, who
should sit up on the edge of the bed or in a chair, if possible.
• The Levine collection tube is inserted through a nostril, and the patient is instructed to swallow the tube.
• When the tube has been fully inserted, a syringe is attached to the end of the tube and filtered distilled water is injected into the
tube.
• The syringe is then used to withdraw 5 to 10 mL of gastric secretions, which is expelled slowly down the sides of the 50-mL
conical collecting tube. Samples should be adjusted to a neutral pH.
• The laboratory may choose to provide sterile receptacles containing 100 mg of sodium carbonate to reduce the acidity; this
improves the recovery of organisms.
• The top of the collection tube is screwed on tightly, and the tube is held upright during prompt delivery to the laboratory.
• Three specimens should be collected over a period of consecutive days.
• Specimens should be processed within 4 hours. Bronchial lavages, washings, and brushings are collected and submitted by
medical personnel.
• These are the specimens of choice for detecting nontuberculous mycobacteria and other opportunistic pathogens in patients
with immune dysfunction.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
Urine Specimens
• The incidence of urogenital infections shows little evidence of decreasing.
• About 2% to 3% of patients with pulmonary tuberculosis show urinary tract involvement, but 30% to 40%
of patients with genitourinary disease have tuberculosis at some other site.
• The clinical manifestations of urinary tuberculosis, which are variable, include frequency of urination (most
common), dysuria, hematuria, and flank pain.
• Definitive diagnosis requires recovery of acid-fast bacilli from the urine.
• Early morning voided urine specimens (40 mL minimum) in sterile containers should be submitted daily
for at least 3 days.
• The collection procedure is the same as for collecting a clean-catch midstream urine specimen.
• The 24-hour urine specimen is undesirable because of excessive dilution, higher contamination, and
difficulty in concentrating.
• Catheterization should be used only if a midstream voided specimen cannot be collected.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
Fecal Specimens
• Acid-fast staining or culture of stool (or both) from patients with AIDS has been used to identify
patients who may be at risk for developing disseminated M. avium complex disease.
• The clinical utility of this practice remains controversial; however, if screening stains and/ or cultures
are positive, dissemination often follows.
• Feces should be submitted in a clean, dry, wax-free container without preservative or diluent.
Contamination with urine should be avoided.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
Tissue and Body Fluid Specimens
• Tuberculous meningitis is uncommon but occurs in both immunocompetent and immunosuppressed
patients.
• A sufficient quantity of specimen is crucial for isolation of acid-fast bacilli from CSF.
• Very few organisms may be present in the spinal fluid, which makes their detection difficult
• At least 10 mL of CSF is recommended for recovery of mycobacteria.
• as much as possible of other body fluids (10 to 15 mL minimum), such as pleural, peritoneal, and
pericardial fluids, should be collected in a sterile container or syringe with a Luer-tip cap.
• Tissues may be immersed in saline or wrapped in gauze.
• Swabs are discouraged, because the recovery of organisms is decreased.
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
Blood Specimens
• Immunocompromised patients, particularly those infected with HIV, can have disseminated mycobacterial infection; most of these
infections are caused by M. avium complex.
• A blood culture positive for MAC is always associated with clinical evidence of disease.
• Recovery of mycobacteria is improved with blood collection in either a broth or the Isolator lysis-centrifugation system.
• Some studies have indicated that the lysis centrifugation system is advantageous, because quantitative data can be obtained with
each blood culture; in patients with AIDS, quantitation of such organisms can be used to monitor therapy and determine the
prognosis.
• However, the necessity of quantitative blood cultures remains unclear.
• Blood for culture of mycobacteria should be collected as for routine blood cultures.
• Blood collected in regular phlebotomy procedures in anticoagulants such as sodium polyanethol sulfonate (SPS), heparin, and
citrate may be used to inoculate cultures for the recovery of Mycobacterium species.
• Conventional blood culture collection systems are unacceptable for the isolation of Mycobacterium spp.
•
specialized automated systems are available for growth of Mycobacterium spp., including the Bactec MGIT 960 system (BectonDickinson, Franklin Lakes, N.J.), and the BacT/ALERT 3D (Biomerieux, Durham, N.C.).
LABORATORY DIAGNOSIS OF
MYCOBACTERIAL INFECTIONS
Wounds, Skin Lesions, and Aspirates
• An aspirate is the best type of specimen for culturing of a skin lesion or wound.
• The skin should be cleansed with alcohol before aspiration of the material into a syringe.
• If the volume is insufficient for aspiration, pus and exudates may be obtained on a swab and then
placed in a transport medium, such as Amie’s or Stuart’s medium (dry swabs are unacceptable).
• a negative culture of a specimen obtained on a swab is not considered reliable, and this should be
noted in the culture report.
SPECIMEN PROCESSING
• Processing to recover acid-fast bacilli from clinical specimens involves several complex
steps, each of which must be carried out with precision.
• Specimens from sterile sites can be inoculated directly to media (small volume) or
concentrated to reduce volume.
• Other specimens require decontamination and concentration.
SPECIMEN PROCESSING
Contaminated Specimens
•
Most specimens submitted for mycobacterial culture consist of organic debris
•
mucin
•
Tissue
•
Serum
•
other proteinaceous material contaminated with organisms
•
sputum
•
Laboratories must process these specimens to kill or reduce contaminating bacteria that can rapidly outgrow mycobacteria, and
mycobacteria are released from mucin and/or cells
•
After decontamination, mycobacteria are concentrated, usually by centrifugation, to enhance their detection by acid-fast stain and culture.
•
Unfortunately, there is no single ideal method for decontaminating and digesting clinical specimens.
•
Rapidly growing mycobacteria are especially susceptible to high or prolonged exposure to greater than or equal to 2% sodium hydroxide
(NaOH).
•
Digestion-decontamination procedures should be as gentle as possible.
SPECIMEN PROCESSING
Inadequate Specimens and Rejection Criteria
• Identification and detection of Mycobacterium spp. is costly and time consuming.
• Specimens should be rejected according to the following guidelines:
• (1) insufficient volume
• (2) contamination with saliva
• (3) dried swabs
• (4) pooled sputum or urine
• (5) container has been compromised, broken or leaking
• (6) length of time from collection to processing is too long.
SPECIMEN PROCESSING
Overview.
• Commonly used digestion-decontamination methods are the
• NaOH method,
• Zephiran-trisodium phosphate method
• N-acetyl-L-cysteine (NALC)– 2% NaOH method.
SPECIMEN PROCESSING
• Another decontaminating procedure that uses oxalic acid is very useful for treating
specimens known to harbor gram-negative rods, particularly Pseudomonas and Proteus
spp., which are extremely troublesome contaminants.
• oxalic acid, NaOH, and mild hydrogen chloride (HCl) may reduce the recovery of M.
ulcerans
• NaOH, a commonly used decontaminant that is also mucolytic, should be used with
caution
• It not only reduces contamination, but also reduces recovery of Mycobacterium spp. as
alkalinity increases, temperature rises, and exposure time increases.
• The sample should be homogenized by centrifugal swirling, minimizing physical agitation.
• The container then should be allowed to sit for 15 minutes so that aerosolized droplets
can fall to the bottom, thus reducing the risk of infection for the laboratory professional.
SPECIMEN PROCESSING
• Several agents can be used to liquefy a clinical specimen:
• N-acetyl-L-cysteine (NALC)
• dithiothreitol (sputolysin)
• enzymes.
• None of these agents are inhibitory to bacterial cells.
• In most procedures, liquefaction (release of the organisms from mucin or cells) is enhanced by vigorous mixing with a
vortex-type mixer in a closed container.
• After mixing as previously described, the container should be allowed to stand for 15 minutes before opening, to
prevent the dispersion of fine aerosols generated during mixing.
• Of utmost importance during processing is strict adherence to processing and laboratory safety protocols.
• All of these procedures should be carried out in a biologic safety cabinet (BSC).
• After digestion and decontamination, specimens are concentrated by centrifugation at greater than or equal to 3000Å~
g.
SPECIMEN PROCESSING
Special Considerations.
• Many specimen types besides respiratory samples contain normal flora and
require decontamination and concentration.
• Aerosol-induced sputum should be treated as sputum.
• Gastric lavages should be processed within 4 hours of collection or neutralized
with 10% sodium carbonate (check with pH paper to make sure the specimen is
at neutral pH) and refrigerated until processed as for sputum.
• If more than 10 mL of watery-appearing aspirate was obtained, the specimen can
be centrifuged at 3600Å~ g for 30 minutes, the supernatant decanted, and the
sediment processed as for sputum.
SPECIMEN PROCESSING
• Urine specimens should be divided into a maximum of four 50-mL centrifuge tubes and centrifuged at 3600Å~ g for 30
minutes.
• The supernatant should be decanted, leaving approximately 2 mL of sediment in each tube.
• The tubes are vortexed to suspend the sediments, and sediments are combined.
• If necessary, distilled water can be added to a total volume of 10 mL.
• This urine concentrate is treated as for sputum or with the sputolysin– oxalic acid method.
• For fecal specimens, approximately 0.2 g of stool (a portion about the size of a pea) is emulsified in 11 mL of sterile,
filtered, distilled water.
• The suspension is vortexed thoroughly, and particulate matter is allowed to settle for 15 minutes.
• Ten milliliters of the supernatant is then transferred to a 50-mL conical centrifuge tube and decontaminated using the
oxalic acid or NALCNaOH method.
SPECIMEN PROCESSING
• Swabs and wound aspirates should be transferred to a sterile, 50-mL conical centrifuge tube
containing a liquid medium (Middlebrook 7H9, Dubos Tween albumin broth) at a ratio of 1 part
specimen to 5 to 10 parts liquid medium.
• The specimen is vortexed vigorously and allowed to stand for 20 minutes.
• The swab is removed, and the resulting suspension is processed as for sputum.
• Large pieces of tissue should be finely minced with a sterile scalpel and scissors.
• This material is homogenized in a sterile tissue grinder with a small amount of sterile saline (0.85%)
or sterile 0.2% bovine albumin; the suspension then is processed as for sputum.
• If the tissue is not known to be sterile, it is homogenized, and half is directly inoculated to solid and
liquid media.
• The other half is processed as for sputum. If the tissue is collected aseptically (i.e., it is sterile), it
may be processed without being treated with NALC-NaOH.
SPECIMEN PROCESSING
Specimens Not Requiring Decontamination
• Tissues or body fluids collected aseptically usually do not require the digestion and decontamination
methods used with contaminated specimens.
• The processing of clinical specimens that do not routinely require decontamination for acid-fast
culture is described here.
• If such a specimen appears contaminated because of color, cloudiness, or foul odor, Gram staining is
performed to detect bacteria other than acid-fast bacilli.
• CSF should be handled aseptically and centrifuged for 30 minutes at 3600Å~ g to concentrate the bacteria.
• The supernatant is decanted, and the sediment is vortexed thoroughly before the smear is prepared and the media
inoculated.
• If insufficient quantity of spinal fluid is received, the specimen should be used directly for smear and culture.
• Recovery of acid-fast bacilli from CSF is difficult, and additional solid or liquid media should be inoculated if material is
available.
• Pleural fluid should be collected in sterile anticoagulant (1 mg/mL ethylenediaminetetraacetic acid [EDTA] or 0.1 mg/mL
heparin).
• If the fluid becomes clotted, it should be liquefied with an equal volume of sputolysin and vigorously mixed.
• To lower the specific gravity and density of pleural fluid, 20 mL is transferred to a sterile, 50-mL centrifuge tube, and the
specimen is diluted by filling the tube with distilled water.
• The tube is inverted several times to mix the suspension and then centrifuged at 3600Å~ g for 30 minutes.
• The supernatant should be removed, and the sediment should be suspended for smear and culture.
SPECIMEN PROCESSING
• Joint fluid and other sterile exudates can be handled aseptically and inoculated directly to media.
• Bone marrow aspirates may be injected into Pediatric Isolator tubes (Alere, Waltham, MA), which
help prevent clotting; the specimen can be removed with a needle and syringe for preparation of
smears and cultures.
• As an alternative, these specimens are either inoculated directly to media or, if clotted, treated with
sputolysin or glass beads and distilled water before concentration.
DIRECT DETECTION METHODS
Microscopy
• Microscopy is considered a reasonably sensitive and rapid
procedure for the presumptive identification of Mycobacterium spp.
in clinical specimens.
DIRECT DETECTION METHODS
Acid-Fast Stains
• The cell walls of mycobacteria contain long-chain, multiply cross-linked fatty acids, called mycolic
acids.
• Mycolic acids probably complex basic dyes, contributing to the characteristic of acid-fastness that
distinguishes mycobacteria from other bacteria.
• Mycobacteria are not the only group with this unique feature.
• Species of Nocardia and Rhodococcus are also partially acid-fast; Legionella micdadei, a causative
agent in pneumonia, is partially acid-fast in tissue.
• Cysts of the genera Cryptosporidium and Isospora are distinctly acid-fast.
• The mycolic acids and lipids in the mycobacterial cell wall probably account for the unusual
resistance of these organisms to the effects of drying and harsh decontaminating agents in addition
to the property of acid-fastness.
DIRECT DETECTION METHODS
• When Gram stained, mycobacteria usually appear as slender, poorly stained, beaded, grampositive bacilli sometimes they appear as “gram neutral,” or “gram-ghosts,” by failing to
take up either crystal violet or safranin.
• Acid-fastness is affected by the age of colonies, the medium on which growth occurs, and
exposure to ultraviolet light.
• Rapidly growing species appear to be acid-fast variable.
• Three types of staining procedures are used in the laboratory for rapid detection and
confirmation of acid fast bacilli:
• fluorochrome,
• Ziehl-Neelsen
• Kinyoun.
DIRECT DETECTION METHODS
• Visualization of acid-fast bacilli in sputum or other clinical material should be considered
only presumptive evidence of tuberculosis, because staining does not specifically identify
M. tuberculosis.
DIRECT DETECTION METHODS
Methods
Fluorochrome Stain.
• Fluorochrome staining is the screening procedure recommended for
laboratories that have a fluorescent (ultraviolet) microscope
• Fluorochrome stain is more sensitive than the conventional carbolfuchsin
stains, because the fluorescent bacilli stand out brightly against the
background
• Because the smear can be examined initially at lower magnifications
(Å~250 to Å~400), more fields can be visualized in a short period.
DIRECT DETECTION METHODS
• In addition, a positive fluorescent smear may be restained using the conventional Ziehl-Neelsen or
Kinyoun procedure, thereby saving the time needed to make a fresh smear.
• Screening of specimens with rhodamine or rhodamine-auramine results in a higher yield of positive
smears and substantially reduces the time needed to examine smears.
• One drawback of the fluorochrome stains is that many rapid-growers may not appear fluorescent
with these reagents.
• All positive fluorescent smears should be confirmed with a Ziehl-Neelsen stain or by examination
by another technologist.
• It is important to wipe the immersion oil from the objective lens after examining a positive smear,
because stained bacilli can float off the slide into the oil, possibly contributing to a false-positive
reading for the next smear examined.
DIRECT DETECTION METHODS
Fuchsin Acid-Fast Stains
Classic carbolfuchsin stain (Ziehl-Neelsen)
• requires heating of the slide for better penetration of the stain into the mycobacterial cell wall;
• it is also known as the hot stain procedure
• Ziehl- Neelsen staining:
• Mycobacterium spp. appear red or have nonmycobacteria appear blue
• Kinyoun acid-fast stain
• similar to Ziehl-Neelsen staining, but no heat is used
• this technique is known as the cold stain procedure
• If present, typical acid-fast bacilli appear as purple to red, slightly curved, short or long rods (2 to 8 μm); they also may
appear beaded or banded (M. kansasii).
• For some nontuberculous species, such as M. avium complex, they appear pleomorphic, usually coccoid.
DIRECT DETECTION METHODS
Examination, Interpretation, and Reporting of Smears
• Before a smear is reported as negative, it should be examined carefully by scanning at least 300 oil
immersion fields (magnification Å~1000), equivalent to three full horizontal sweeps of a smear that
is 2 cm long and 1 cm wide.
• Because the fluorescent stain can be examined using a lower magnification (Å~250 or Å~450) than
that required for a fuchsin-stained smear, the equivalent number of fields (30) can be examined in
less time, which makes the fluorochrome stain the preferred method.
• When acid-fast organisms are observed on a smear, the report should include information about the
type of staining method used and the quantity of organisms.
• The overall sensitivity of an acid-fast smear ranges from 20% to 80%.
• factors that can infuence acid fast smear sensitivity:
• specimen type
• staining method
• culture method
• However, cross contamination of slides during the staining process and use of water contaminated
with saprophytic mycobacteria can lead to false-positive results.
• Staining receptacles should not be used; acid-fast bacilli can also be transferred from one slide to
another in immersion oil.
• because of its simplicity and speed, the stained smear is an important and useful test, particularly for
detection of smear-positive patients (“infectious reservoirs”), who pose the greatest risk to others
in their environment.
DIRECT DETECTION METHODS
Antigen-Protein Detection
• The detection of microbial products or components
• tuberculostearic acid is a fatty acid that can be extracted from the cell wall of mycobacteria and detected by gas
chromatography or mass spectrometry in clinical samples containing few mycobacteria
• Because of the limited number of species that can cause meningitis and because M. tuberculosis appears to be the only one of
these species that releases tuberculostearic acid into the surrounding environment, the presence of this substance in CSF is
thought to be diagnostic of tuberculous meningitis
• Performance of this assay is limited to a few laboratories.Various immunoassays for antigen detection directly in clinical
specimens, including sputum and CSF, have been evaluated and show some promise.
• Production of adenosine deaminase, a host enzyme, is increased in certain infections caused by M. tuberculosis.
• For example, elevated levels of this enzyme were found in most patients with tuberculous pleural effusions (98% sensitive); the
test for the enzyme also was determined to be highly specific (96% specificity).
DIRECT DETECTION METHODS
Immunodiagnostic Testing
• interferon-gamma release assays have become more widely used for the diagnosis of tuberculosis.
• T-SPOT-TB (Oxford Immunotec, Oxford, United Kingdom) and QuantiFERON
•
The T-SPOT-TB assay is an enzymelinked immunospot assay that requires isolation and incubation of peripheral blood
mononuclear cells (PBMCs).
• Gold In-Tube (QFNG-IT; Cellestis, Chadstone,Victoria, Australia), do not typically cross react with
nontuberculous mycobacterium, are not affected by the BCG vaccine, and are not as variable as the
historical serologic tuberculin skin tests.
•
The QFNG-IT assay measures the stimulation of T-cell interferon-gamma in whole blood in a tube precoated with M.
tuberculosis antigens.
•
It yields results in approximately 8 hours.
• Neither assay distinguishes between latent and active infections.
• In addition, specificity and sensitivity vary in the population tested, including immunocompromise patients
and children.
DIRECT DETECTION METHODS
Genetic Sequencing and Nucleic Acid Amplification
• PCR-based sequencing for mycobacterial identification consists of PCR amplification of mycobacterial
DNA with genusspecific primers and sequencing of the amplicons.
• The organism is identified by comparison of the nucleotide sequence with reference sequences
• Despite the accuracy of PCR-based sequencing to identify mycobacteria, problems remain: the sequences
in some databases are not accurate
• The limitation of this sequence is that it is not a genus-specific sequence; therefore, results may be
affected by contaminating bacteria.
• conventional and real-time PCR, have been used to detect M. tuberculosis directly in clinical specimens
• Amplicor Mycobacterium tuberculosis test (Roche Diagnostic Systems, Branchburg, New Jersey) uses PCR to detect
M. tuberculosis directly in respiratory specimens
• Amplified Mycobacterium tuberculosis Direct Test (AMTD; Gen-Probe, San Diego, California) is based on ribosomal
RNA amplification
DIRECT DETECTION METHODS
DNA Microarrays
• DNA microarrays are also attractive for rapid examination of large
numbers of DNA sequences by a single hybridization step.
• This approach has been used to simultaneously identify mycobacterial
species and detect mutations that confer rifampin resistance in
mycobacteria.
• Fluorescent-labeled PCR amplicons generated from bacterial colonies are
hybridized to a DNA array containing nucleotide probes.
DIRECT DETECTION METHODS
Chromatographic Analysis
• Analysis of mycobacterial lipids by chromatographic methods:
• thin-layer chromatography
• gas-liquid chromatography (GLC)
• capillary gas chromographic methods
• reverse-phase high-performance liquid chromatography (HPLC)
DIRECT DETECTION METHODS
• In HPLC, a liquid mobile phase is combined with various technical advances to separate large
cellular metabolites and components.
• HPLC of extracted mycobacteria is a specific and rapid method for identifying species. Many state
health departments and the CDC now use this method routinely.
• The long-chain mycolic acids are separated better by HPLC than by GLC, because they do not
withstand the high temperatures needed for GLC.
• The patterns produced by different species are very easily reproducible, and a typical identification
requires only a few hours
DIRECT DETECTION METHODS
Cultivation
• A combination of different culture media is required to optimize recovery of mycobacteria from
culture; at least one solid medium in addition to a liquid medium should be used.
• The ideal media combination should be economical and should support the most rapid and
abundant growth of mycobacteria, allow for the study of colony morphology and pigment
production, inhibit the growth of contaminants.
DIRECT DETECTION METHODS
Solid Media
• Solid media, are recommended because of the development of characteristic, reproducible colonial morphology,
good growth from small inocula, and a low rate of contamination.
• Optimally, at least two solid media (a serum [albumin] agar base medium, [e.g., Middlebrook 7H10] and an eggpotato base medium [e.g., Löwenstein-Jensen, or L-J]) should be used for each specimen (these media are
available from commercial sources).
• All specimens must be processed appropriately before inoculation. It is imperative to inoculate test organisms to
commercially available products for quality control
DIRECT DETECTION METHODS
• Cultures are incubated at 35° C in the dark in an atmosphere of 5% to 10% carbon
dioxide (CO2) and high humidity.
• Tube media are incubated in a slanted position with screw caps loose for at least 1 week
to allow for evaporation of excess fluid and the entry of CO2; plated media are either
placed in a CO2-permeable plastic bag or wrapped with CO2-permeable tape.
• If specimens obtained from the skin or superficial lesions are suspected to contain M.
marinum or M. ulcerans, an additional set of solid media should be inoculated and
incubated at 25° to 30° C.
• a chocolate agar plate (or placement of an X-factor [hemin] disk on conventional media)
and incubation at 25° to 33° C is needed for recovery of M. haemophilum from these
specimens. RGM optimally require incubation at 28° to 30° C.
M. TUBERCULOSIS COLONIES ON
L . W E N S T E I N - J E N S E N AG A R A F T E R
8 W E E K S O F I N C U B AT I O N .
A DIFFERENT COLONIAL
M O R P H O L O G Y I S S E E N O N C U LT U R E
O F O N E S T R A I N O F M . AV I U M
COMPLEX.
C , M. KANSASII COLONIES
EXPOSED TO LIGHT
SCOTOCH ROMOG EN
M. G OR DONAE SH OW I NG
Y E L L OW COL ONI E S
S M O OT H , M U LT I L O B AT E C O L O N I E S
O F M . F O RT U I T U M O N L . W E N S T E I N JENSEN MEDIUM
DIRECT DETECTION METHODS
• Cultures are examined weekly for growth.
• Contaminated cultures are discarded and reported as “contaminated, unable to detect
presence of mycobacteria”; additional specimens are also requested.
• If available, sediment may be recultured after enhanced decontamination or by inoculating
the sediment to a more selective medium. Most isolates appear between 3 and 6 weeks; a
few isolates appear after 7 or 8 weeks of incubation.
• When growth appears, the rate of growth, pigmentation, and colonial morphology are
recorded.
• After 8 weeks of incubation, negative cultures (those showing no growth) are reported,
and the cultures are discarded.
DIRECT DETECTION METHODS
Liquid Media
• In general, use of a liquid media system reduces the turnaround time for isolation of acid-fast bacilli to
approximately 10 days, compared with 17 days or longer for conventional solid media.
• Several different systems are available for culturing and detecting the growth of mycobacteria in liquid
media.
• Growth of mycobacteria in liquid media, regardless of the type, requires 5% to 10% CO2; CO2 is either
already provided in the culture vials or is added according to the manufacturer’s instructions.
• When growth is detected in a liquid medium, acid-fast staining of a culture aliquot is performed to
confirm the presence of acid-fast bacilli, and the material is subcultured to solid agar. G
• ram staining can also be performed if contamination is suspected.
DIRECT DETECTION METHODS
Interpretation
• Although isolation of MAC organisms indicates infection, the clinician must determine the clinical
significance of isolating NTM in most cases; in other words, does the organism represent mere
colonization or significant infection?
• Because these organisms vary greatly in their pathogenic potential, can colonize an individual
without causing infection, and are ubiquitous in the environment, interpretation of a positive NTM
culture is complicated.
• Therefore, the American Thoracic Society has recommended diagnostic criteria for NTM disease to
help physicians interpret culture results.
DIRECT DETECTION METHODS
Conventional Phenotypic Tests
• Growth Characteristics. Preliminary identification of mycobacterial isolates depends on the organisms’
rate of growth, colonial morphology,colonial texture, pigmentation and, in some instances, the permissive
incubation temperatures of mycobacteria.
• Despite the limitations of phenotypic tests, the mycobacterial growth characteristics are helpful for
determining a preliminary identification (e.g., an isolate appears as rapidly growing mycobacteria). To
perform identification procedures, quality control organisms should be tested along with unknowns.
• The commonly used quality control organisms can be maintained in broth at room temperature and
transferred monthly.
• In this way they are always be available for inoculation to test media along with suspensions of the
unknown mycobacteria being tested.
DIRECT DETECTION METHODS
Growth Rate.
• The rate of growth is an important criterion for determining the initial category of an isolate.
• Rapid-growers usually produce colonies within 3 to 4 days after subculture.
• However, even a rapid-grower may take longer than 7 days to initially produce colonies because of inhibition
by a harsh decontaminating procedure.
• Therefore, the growth rate (and pigment production) must be determined by subculture (
• The dilution of the organism used to assess the growth rate is critical.
• Even slow-growing mycobacteria appear to produce colonies in less than 7 days if the inoculum is too heavy.
One organism particularly likely to exhibit false-positive rapid growth is M. flavescens.
• This species therefore serves as an excellent quality control organism for this procedure.
DIRECT DETECTION METHODS
Pigment Production.
• mycobacteria may be categorized into three groups based on pigment production. P
• To achieve optimum photochromogenicity, colonies should be young, actively metabolizing, isolated, and
well aerated.
• Although some species (e.g., M. kansasii) turn yellow after a few hours of light exposure, others (e.g., M.
simiae) may take prolonged exposure to light.
• Scotochromogens produce pigmented colonies even in the absence of light, and colonies often become
darker with prolonged exposure to light
• One member of this group, M. szulgai, is peculiar in that it is a scotochromogen at 35° C and
nonpigmented when grown at 25° to 30° C.
• all pigmented colonies should be subcultured to test for photoactivated pigment at both 35° C and 25° to
30° C.
• Nonchromogens are not affected by light.
DIRECT DETECTION METHODS
Biochemical Testing.
• Once categorized into a preliminary subgroup based on its growth characteristics, an organism must be definitively
identified to species or complex level.
• Although conventional biochemical tests can be used for this purpose, new methods (discussed later in this section)
have replaced biochemical tests for identifying mycobacterial species because of the previously discussed limitations of
phenotypic testing.
• Although key biochemical tests are still discussed in this edition, the reader must be aware that this approach to
identification ultimately will be replaced by molecular methods.
DIRECT DETECTION METHODS
Niacin.
• Niacin (nicotinic acid) plays an important role in the oxidation-reduction reactions that occur during mycobacterial
metabolism.
• Although all species produce nicotinic acid, M. tuberculosis accumulates the largest amount. (M. simiae and some strains
of M. chelonae also produce niacin.)
• Niacin therefore accumulates in the medium in which these organisms are growing.
• A positive niacin test is preliminary evidence that an organism that exhibits a buff-colored, slow-growing, rough colony
may be M. tuberculosis
• ,this test is not sufficient to confirm identification.
• If sufficient growth is present on an initial L-J slant (the egg-base medium enhances accumulation of free niacin), a niacin
test can be performed immediately.
• If growth on the initial culture is scant, the subculture used for growth rate determination can be used. If this culture
yields only rare colonies, the colonies should be spread around with a sterile cotton swab (after the growth rate has
been determined) to distribute the inoculum over the entire slant.
DIRECT DETECTION METHODS
• The slant then is incubated until light growth over the surface of the medium is visible.
• For reliable results, the niacin test should be performed only from cultures on L-J medium that are
at least 3 weeks old and show at least 50 colonies; otherwise, enough detectable niacin might not
have been produced.
DIRECT DETECTION METHODS
Nitrate Reduction.
• This test is valuable for identifying M. tuberculosis, M. kansasii, M. szulgai, and M. fortuitum.
• The ability of acid-fast bacilli to reduce nitrate is influenced by the age of the colonies, temperature, pH, and enzyme
inhibitors.
• Although rapid-growers can be tested within 2 weeks, slow-growers should be tested after 3 to 4 weeks of luxuriant
growth.
• Commercially available nitrate strips yield acceptable results only with strongly nitrate-positive organisms, such as M.
tuberculosis. This test may be tried first because of its ease of performance.
• The M. tuberculosis–positive control must be strongly positive in the strip test, or the test results are unreliable.
• If the paper strip test is negative or if the control test result is not strongly positive, the chemical procedure must be
carried out using strong and weakly positive controls.
DIRECT DETECTION METHODS
Catalase
• Most species of mycobacteria, except for certain strains of M. tuberculosis complex (some isoniazidresistant strains) and
M. gastri, produce the intracellular enzyme catalase, which splits hydrogen peroxide into water and oxygen.
• Catalase can be assessed by using the semiquantitative catalase test or the heat-stable catalase test.
• • The semiquantitative catalase test is based on the relative activity of the enzyme, as determined by the height of a
column of bubbles of oxygen formed by the action of untreated enzyme produced by the organism.
• Based on the semiquantitative catalase test, mycobacteria are divided into two groups: those that produce less than 45
mm of bubbles and those that produce more than 45 mm of bubbles.
• The heat-stable catalase test is based on the ability of the catalase enzyme to remain active after heating (i.e., it is a
measure of the enzyme’s heat stability).
• When heated to 68° C for 20 minutes, the catalase of M. tuberculosis, M. bovis, M. gastri, and M. haemophilum becomes
inactivated.
DIRECT DETECTION METHODS
Tween 80 Hydrolysis.
• The commonly nonpathogenic, slow-growing scotochromogens and nonphotochromogens produce
a lipase that can hydrolyze Tween 80 (the detergent polyoxyethylene sorbitan monooleate) into
oleic acid and polyoxyethylated sorbitol, whereas pathogenic species do not.
• Tween 80 hydrolysis is useful for differentiating species of photochromogens, nonchromogens, and
scotochromogens.
• Because laboratory prepared media have a very short shelf life, the CDC recommends use of a
commercial Tween 80 hydrolysis substrate (Becton-Dickinson, Franklin Lakes, N.J. or Remel
Laboratories, Lenexa, Kansas) that is stable for up to 1 year.
DIRECT DETECTION METHODS
Tellurite Reduction.
• Some species of mycobacteria reduce potassium tellurite at variable rates.
• The ability to reduce tellurite in 3 to 4 days distinguishes members of MAC from most other
nonchromogenic species.
• All rapid-growers reduce tellurite in 3 days.
DIRECT DETECTION METHODS
• Arylsulfatase.
• The enzyme arylsulfatase is present in most mycobacteria. Test conditions can be varied to
differentiate different forms of the enzyme.
• The rate at which this enzyme breaks down phenolphthalein disulfate into phenolphthalein (which
forms a red color in the presence of sodium bicarbonate) and other salts helps to differentiate
certain strains of mycobacteria.
• The 3-day test is particularly useful for identifying the potentially pathogenic rapid-growers M.
fortuitum and M. chelonae.
• Slow-growing M. marinum and M. szulgai are positive in the 14-day test.
DIRECT DETECTION METHODS
• Growth Inhibition by Thiophene-2-Carboxylic Acid Hydrazide (TCH). This test is used to distinguish
M. bovis from M. tuberculosis, because only M. bovis is unable to grow in the presence of 10 mg/mL
of TCH.
DIRECT DETECTION METHODS
Other Tests
• Other tests are often performed to make more subtle distinctions between species
• However, performing all the procedures necessary for definitive identification of mycobacteria is not
cost-effective for routine clinical microbiology laboratories; therefore, specimens that require
further testing can be forwarded to regional laboratories.
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
• Drug-resistant tuberculosis is a major health threat; more than 500,000 cases of multidrug-resistant
(MDR) tuberculosis occur each year.
• Multidrug-resistant tuberculosis is resistant to rifampin and isoniazid, the two drugs most often used
as effective treatment against tuberculosis.
• strains of extensively drug-resistant tuberculosis (XDR TB) are emerging that are resistant not only
to rifampin and isoniazid, but also to quinolones and other drugs, such as aminoglycosides and
capreomycin.
• Standardized methods for susceptibility testing, including direct and indirect testing and new
molecular tools, currently are available for susceptibility testing.
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
M.TUBERCULOSIS COMPLEX
• In vitro drug susceptibility testing should be performed on the first isolate of M. tuberculosis from
all patients.
• Susceptibility testing of M. tuberculosis requires meticulous care in the preparation of the medium,
selection of adequate samples of colonies, standardization of the inoculum, use of appropriate
controls, and interpretation of results.
• Laboratories that see very few positive cultures should consider sending isolates to a reference
laboratory for testing. Isolates must be saved in sterile 10% skim milk in distilled water at −70° C
for possible future additional studies (e.g., susceptibilities if the patient does not respond well to
treatment).
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
Direct Versus Indirect Susceptibility Testing
• Susceptibility tests may be performed by either the direct or indirect method. The direct method
uses as the inoculum a smear-positive concentrate containing more than 50 acid-fast bacilli per 100
oil immersion fields; the indirect method uses a culture as the inoculum source.
• Although direct testing provides more rapid results, it is less standardized, and contamination may
occur.
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
Conventional Methods
•
The development of primary drug resistance in tuberculosis represents an increase in the proportion of resistant organisms.
•
This increase in resistant organisms results from a spontaneous mutation and subsequent selection to predominance of these drugresistant mutants by the action of a single or ineffective drug therapy.
•
A poor clinical outcome is predicted with an agent when more than 1% of bacilli in the test population are resistant. If an isolate is
reported as resistant to a drug, treatment failure is likely if this drug is used for therapy.
•
Drug resistance is defined for M. tuberculosis complex in terms of the critical concentration of the drug. The critical concentration of a
drug is the amount of drug required to prevent growth above the 1% threshold of the test population of tubercle bacilli.
•
Four general methods are used throughout the world to determine the susceptibility of M. tuberculosis isolates to various antituberculous
agents.
•
Initial isolates of M. tuberculosis are tested against five antimicrobials, which are referred to as primary drugs
•
If resistance to any of the primary drugs is detected, a second battery of agents is tested (Box 43-4).
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
Therapy
• Therapy directed against M. tuberculosis depends on the susceptibility of the isolate to various antimicrobial agents.
• To prevent the selection of resistant mutants, treatment of tuberculosis requires four drugs:
•
Isoniazid
•
Rifampin
•
Ethambutol
•
Pyrazinamide
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
• Initial therapy includes all four drugs for 8 weeks.
• However, if drug susceptibility is determined for isoniazid, rifampin, and pyrazinamide, ethambutol may be
discontinued.
• This is the preferred therapy for initial treatment, followed by isoniazid and rifampin for an additional 18
weeks.
• The most common two-drug regimen is isoniazid (INH, also known as isonicotinylhydrazine) and rifampin.
• The combination is administered for 9 months in cases of uncomplicated tuberculosis; if pyrazinamide is
added to this regimen during the first 2 months, the total duration of therapy can be shortened to 6 months.
Ethambutol may also be added to the regimen.
• INH prophylaxis is recommended for individuals with a recent skin test conversion who are disease free.
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
NONTUBERCULOUS MYCOBACTERIA
•
In general, the treatment of patients infected with NTM requires more individualization of therapy than does the treatment of patients
with tuberculosis.
•
This individualization is based on the species of mycobacteria recovered, the site and severity of infection, antimicrobial drug susceptibility
results, concurrent diseases, and the patient’s general condition.
•
Currently, sufficient data exist to allow general recommendations for susceptibility testing of MAC, M. kansasii, and M. marinum.
•
Pulmonary infections with M. avium complex are often treated with clarithromycin, rifampin, and ethambutol (or streptomycin or amikacin
for severe disease). If the infection is disseminated, clarithromycin, ethambutol, and rifabutin may be prescribed.
•
Pulmonary infections with M. kansasii are treated with isoniazid, rifampin, and ethambutol.
•
M. marinum skin and soft tissue infections may be treated with either clarithromycin and ethambutol, clarithromycin and rifampin, or
rifampin and ethambutol.
•
Susceptibility testing should be performed on clinically significant, rapidly growing mycobacteria.
•
Skin and soft tissue infections, if susceptible, are treated with clarithromycin and at least one additional drug based on susceptibility testing.
•
Pulmonary infections with M. abscessus should also be treated with a multidrug regimen that includes clarithromycin, if susceptible, and
then additional drugs based on susceptibility testing.
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
Antitubercular Agents Commonly Tested against M. tuberculosis
• Primary Drugs
•
Streptomycin
•
Isoniazid
•
Rifampin
•
Ethambutol
•
Pyrazinamide
ANTIMICROBIAL SUSCEPTIBILITY
TESTING AND THERAPY
•
Secondary Drugs
•
Ethionamide
•
Capreomycin
•
Ciprofloxacin
•
Doxycycline or minocycline
•
Ofloxacin
•
Kanamycin
•
Cycloserine
•
Rifabutin
•
Trimethoprim-sulfamethoxazole
PREVENTION
• As previously mentioned, prophylactic chemotherapy with INH is used when known or suspected
primary tuberculous infection poses a risk of clinical disease.
• At present, the BCG vaccine is the only vaccine available against tuberculosis.
• The effectiveness of this live vaccine is controversial, because studies have demonstrated
ineffectiveness to 80% protection.
• The greatest potential value for this vaccine is in developing countries with high prevalence rates for
tuberculosis.
• At this time, at least four types of antituberculosis vaccines are currently being evaluated in
experimental studies in animals.
SMEAR O F A SPUTUM SPECIMEN SH OW S
AC I D - FA S T B AC I L L I ( M Y C O B AC T E R I U M
T U B E R C U L O S I S ; K I N YO U N , Å ~ 4 0 0 )