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Non-molecular identification of nontuberculous mycobacteria in the clinical microbiology laboratory: What's the real deal?
Clinical Microbiology Newsletter Vol. 28, No. 10
May 15, 2006
Non-Molecular Identification of Nontuberculous Mycobacteria in the Clinical Microbiology Laboratory: What’s the Real Deal? Leslie Hall, M.MSc. and Glenn D. Roberts, Ph.D., Department of Laboratory Medicine and Pathology, Division of Clinical Microbiology, Mayo Clinic and Mayo Clinic College of Medicine, Rochester, Minnesota
Abstract The identification of mycobacteria, other than the Mycobacterium tuberculosis complex, is a challenge for most routine clinical microbiology laboratories. Laboratories may use nucleic acid probe identification of Mycobacterium avium-M. intracellulare, Mycobacterium gordonae, and Mycobacterium kansasii, if financial resources are available. Currently, 109 species of mycobacteria have been described using nucleic acid sequencing. Phenotypic characters are generally incomplete in the literature, and for this reason, it is recommended that routine clinical microbiology laboratories refer cultures to state health laboratories or other reference laboratories for identification.
Introduction In today’s changing world, the clinical microbiology laboratory is faced with numerous challenges, including new technology, expense management, reduced personnel, improving quality, shortened turnaround times, increased costs, and frequent name changes of organisms, among other things. Another challenge of a different type is the identification of mycobacteria. Even though the mycobacteria represent only a small percentage of the organisms seen in the clinical laboratory, they require the greatest amount of time and expertise for recovery and identification. Most routine clinical microbiology laboratories are not experienced enough to offer full mycobacteriology services. The major emphasis in mycobacteriology during recent years has been on the rapid detection and identification of Mycobacterium tuberculosis, due to its
Mailing Address: Dr. Glenn D. Roberts, Division of Clinical Microbiology, Mayo Clinic, Hilton 460C, 200 First Street Southwest, Rochester, MN 55905. Tel.: 507-284-3704. Fax: 507-284-4274. E-mail: [email protected] Clinical Microbiology Newsletter 28:10,2006
public health importance. The Centers for Disease Control and Prevention (CDC) established guidelines for detection and identification of this organism, and every state health laboratory and most laboratories of moderate size are now very familiar with this important pathogen. Molecular methods have enhanced the rapid detection and identification of common mycobacteria, including M. tuberculosis. Unfortunately, smaller clinical microbiology laboratories do not have the necessary resources to accurately identify the mycobacteria and may use a reference laboratory for these services. To complicate matters, there are currently 109 valid species of mycobacteria (1), excluding members of the M. tuberculosis complex and Mycobacterium leprae. Table 1 presents a list of these species and when each was described; many have been associated with human disease, as recently summarized by Tortoliand and Debrunner et al. (2-4). Nucleic acid sequencing was used to identify most of the newly described species, and only a limited number of phenotypic characteristics were published. Interestingly, even © 2006 Elsevier
though each organism has a species name, clinicians and laboratorians often still use the collective term “nontuberculous mycobacteria” to describe the group, while many insist on using the term “atypical mycobacteria.” Just how far should the routine clinical microbiology laboratory go in terms of trying to identify mycobacteria to the species level? Most cannot afford to use currently available molecular methods, perhaps with the exception of nucleic acid probes that have been commercially available for many years. The value of non-molecular testing and recommendations for identification of the species of mycobacteria other than the M. tuberculosis complex will be discussed. In addition, some current thoughts about what these mycobacteria should be named will be included.
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Identification of Mycobacteria Other than M. tuberculosis Complex and M. leprae Identification by colonial morphologic features In 1959, Ernest Runyon (5) developed a classification scheme that placed the common mycobacteria, other than the M. tuberculosis complex, into four groups based on the presence or absence of pigment production: group I, photochromogenic species; group II, scotochromogenic species; group III, nonchromogenic species; and group IV, rapidly growing species. This scheme was helpful to the clinical laboratory when trying to identify the limited number of mycobacteria recovered from clinical specimens at that time. Vestal and Kubica (6), in 1966, described the differential colonial morphologic features of mycobacteria on Middlebrook and Cohn 7H10 agars. Colonies were examined microscopically to determine if they were smooth or rough, pigmented or not, domed or spreading, transparent or not, filamentous or not, and the growth rate was also considered. In 1970, Runyon (7) refined the process and showed that M. tuberculosis, Mycobacterium kansasii, Mycobacterium avium-M. intracellulare, Mycobacterium xenopi, Mycobacterium fortuitum, and Mycobacterium abscessus could be distinguished from each other. He provided illustrative photographs of the colonies of mycobacteria using lowpower microscopy and a limited discussion of the morphologic and biochemical features of all the species; he also included information on Mycobacterium ulcerans, Mycobacterium scrofulaceum, and Mycobacterium marinum. In 1993, Welch et al. (8) reintroduced the microscopic observation of mycobacterial colonies, although this time the emphasis was on the time to
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recovery and identification. These characteristics were helpful for identifying some species, but because the information was not available for many species, some might have had overlapping features. The Runyon grouping provided a useful means of grouping the mycobacteria with similar characteristics, and it is still used in many laboratories today to separate these organisms into these broad categories. However, the groupings are not homogenous, and the distinctions between them are not definitive. Most descriptions are limited to whether a colony is rough or smooth, and this information alone is not helpful, since many of the species share the same features. It should be emphasized that this classification scheme is now largely obsolete due to the large number of species of mycobacteria that have similar morphologic features; it should be used only to broadly define pigmentation characteristics or to describe the growth rate, i.e., rapidly growing species. Pigment production. In general, information related to pigment production of colonies requires that cultures be incubated in the presence and absence of light. Since cultures are not routinely incubated under both conditions, the person who examines the cultures must make a conscious decision as to whether cultures are incubated in the light or dark. If incubators are free from light and doors have been kept closed to prevent exposure of cultures to light, then pigmented colonies may be considered to be scotochromogenic mycobacteria. If organisms are suspected to be photochromogenic, they must be incubated in the presence and absence of light. We reviewed the phenotypic information presented in three recent publications (9-11) and noted that information was available for 68 species (62.4% of described species) of
© 2006 Elsevier
mycobacteria, and of these, only 88.2% described pigment production. Some variation occurs within species; most strains of M. avium-M. intracellulare are nonchromogenic, while others are scotochromogenic. Mycobacterium szulgai is photochromogenic at 25ºC and photochromogenic at 37ºC. As shown in Table 2, there is no consensus on whether Mycobacterium celatum is pigmented. Growth rate The growth rate has been defined by some as the time interval between culturing a clinical specimen and the time that mature colonies are observed. Rapidly growing mycobacteria are said to appear within 7 days, and those that appear later are classified as slowly growing species. There are several inherent problems with this concept. The growth actually depends on a number of factors, including the metabolic rate of an organism, the number of CFU present in the clinical specimen, how often cultures are examined during their incubation, and the medium on which the culture is growing. In reality, a few cells of a rapidly growing species present in a clinical specimen may require more than 7 days for growth, and conversely, a large number of cells of a slowly growing species may require less than 7 days for growth to appear. These are not insignificant problems, and the clinical microbiology laboratory must consider this when using the growth rate as a phenotypic character. Many clinical microbiology laboratories read cultures once weekly, and the exact growth rate cannot be determined. A subculture of an organism that is observed frequently is the best way to determine whether an organism is a rapidly or slowly growing species. However, this adds a significant amount of time to the already slow process of identifying the mycobacteria.
Clinical Microbiology Newsletter 28:10,2006
Acid-fast stain The acid-fast stain is another traditional method that has been used for many years to detect mycobacteria present in clinical specimens; it has also been used to stain cultures to determine whether they have the characteristics of a mycobacterium. The method cannot distinguish one species from another, but it is very important to determine whether an organism is acidfast when trying to place it in the genus Mycobacterium or a related genus.
Table 1. Species of mycobacteria other than members of the M. tuberculosis complex and M. leprae listed by the date they were first described
Optimal growth temperature The optimal growth temperature is a phenotypic character that has been helpful as an aid for the identification of a limited number of mycobacteria. In general, Mycobacterium thermoresistibile grows best at 45ºC, while 42ºC is optimal for M. xenopi. Isolates of M. marinum, Mycobacterium haemophilum, and M. ulcerans prefer temperatures of 30ºC; however, other species share this trait. In general, temperature studies are useful for the separation of only a very limited number of common species.
Identification Using Traditional Biochemical Testing Traditional biochemical tests have been used with some success since the late 1950s and early 1960s to determine the phenotypic characters of some commonly encountered species of mycobacteria. They have been used in combination with colonial morphology, pigment production, and growth rate, along with the acid-fast stain. Representative examples are nitrate reduction, catalase production, catalase inactivation at 68ºC, hydrolysis of Tween, reduction of potassium tellurite, tolerance of 5% NaCl, iron uptake, and arylsulfatase and urease production. A detailed discussion of each test is available in the literature (9,12). Until recently, many of the common species of mycobacteria seen in clinical laboratories were identified using traditional methods. Interestingly, in our experience, biochemical tests failed to provide a definitive identification in many instances, and some were made on a “best-fit” basis when biochemical profiles were considered. Table 2 presents the most common biochemical tests used to aid in the identification of mycobacteria. Clinical Microbiology Newsletter 28:10,2006
Year
Species
Year
Species
1889
M. smegmatis
1987
M. poriferae
1899
M. phlei
1990
1901
M. avium
1912
M. lepraemurium
M. avium spp. paratuberculosis M. avium spp. silvaticum M. cookii
1923
M. chelonae M. paratuberculosis
1992
1926
M. marinum
M. peregrinum M. alvei M. confluentis M. madagascariense
1938
M. fortuitum spp. M. fortuitum
1993
1949
M. intracellulare
1950
M. ulcerans
1953
M. abscessus
M. brumae M. celatum M. genavense M. hiberniae M. intermedium
1955
M. kansasii
1995
1956
M. scrofulaceum
1959
M. xenopi
M. branderi M. interjectum M. mucogenicum
1962
M. flavescens M. gordonae
1996
1964
M. vaccae
M. conspicuum M. lentiflavum M. aurum M. hodleri
1965
M. simiae M. nonchromogenicum M. parafortuitum
1997
1966
M. gastri M. terrae complex M. thermoresistibile
M. hassicum M. mageritense M. triplex M. novocastrense
1998
M. bohemicum M. heidlebergense
1967
M. chitae
1999
1970
M. triviale
1971
M. asiaticum M. duvali M. gilvum M. obuense
M. goodii M. murale M. tusciae M. wolinskyi
2000
M. botniense M. elephantis M. kubicae M. septicum
2001
M. doricum M. fredriksbergense M. heckeshornense M. immunogenum
2002
M. holasaticum M. lacus M. palustre M. vanbaalenii
2003
M. montefiorense M. pinnipedii
2004
M. boenickei M. brisbanense M. canariasense M. chimaera M. cosmeticum M. houstonense M. nebraskense M. neworleansense M. parascrofulaceum M. paramense M. psychrotolerans M. pyrenivorans M. saskatchewanense M. shottsii
1972
M. szulgai M. neoaurum
1973
M. farcinogenes M. senegalense
1974
M. gadium
1977
M. malmoense
1978
M. haemophilum
1979
M. komossense
1980
M. sphagni
1981
M. achiense M. agri M. chubuense M. rhodesiae M. tokaiense
1982
M. shimoidei
1983
M. austroafricanum M. diernhoferi M. fallax M. porcinum M. pulveris
1986
M. fortuitum spp. M. acetamidolyticum M. chlorophenolicum M. moriokaense
© 2006 Elsevier
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Pigmentation
Niacin production
Carboxylic acid hydrazide
Nitrate reduction
Semiquantitative catalase (mm of bubbles)
Inactivation of catalase at 68˚C
Tween 80 hydrolysis
Tellurite reduction
Salt tolerance
Iron uptake
Arylsulfatase
9-11 10,11 9 9 9-11 10 9-11 10,11 9-11 9 10 10,11 9-11 10 10,11 10,11 9 9 10 9 9-11 9-11 9 9, 11 10,11 9-11 9-11 10 10 10,11 9-11 9-11 9-11 9 9 10,11 10,11 9-11 10 9-11 9 9 11 9-11 10,11 11 10,11 10,11 10,11 9 11 9 9 9-11 9,11 10,11 9 9 9 9 9-11 9 9-11 9 9,11 10 9-11 9
Na N P N P P N N NC N P N P P P P P N P N N NC P N P P N P P N N P P N P P N P P N N P ND N P ND P P N P ND P N N N ND P NH P N N N P N P P N NH
NDb N N N N ND N N N NH ND ND ND ND N ND N N ND N N N N N N N N ND ND ND N N NC N N ND N N ND ND N NH N N ND N N ND ND N NH N N N N P NH N N N N N N N N ND N N
ND ND P P ND ND ND ND P P ND ND P ND P N P P ND P P ND P P NH ND ND ND ND P ND P NH P P ND ND P ND P P P ND ND ND P P ND ND P N P ND ND P P P P P P P P P P P ND ND P
N P N N N N N P N n N P N N P P P P P N N P N NC N N N P N P N N N N P NC P N N NC N N N NH N ND P NH P P N N P P NC N N P P NH P P P N P P P N
>45 >45 >45 <45 <45 <45 <45 >45 <45 >45 ND >45 <45 ND <45 >45 >45 >45 ND <45 >45 NC >45 <45 >45 <45 N <45 ND ND ND NC >45 <45 >45 >45 <45 NC ND NH <45 <45 <45 >45 >45§ <45 <45 <45 >45 >45 ND >45 ND ND <45 <45 >45 <45 >45 >45 >45 >45 <45 <45 >45 ND <45 <45
NHc P P NH P N NC NC P NH ND P NC ND NC P P P ND N P NC P N P P P P ND N ND P P NH P ND N NH ND N NH N N N NH NH P P P P ND P NH ND NC NH P N P P P P P P P ND NC NH
NCd P P N N N N P N NH NH N P N N P P NH P P NC NH P N N N NC ND P NH ND NC P N P NC NH N P N P P N P P P NC P NC P ND N ND ND P N N P NH P N P P N P P NH P
ND ND N P ND ND ND NH P P ND ND N ND P N NH P ND NH N NH N N ND ND N ND ND P NH NH NH P NH ND P N ND NH P NH P NC ND NH ND ND ND P ND NH P ND ND ND P P NH NH ND N NH ND P ND NH NH
NC N N N N ND NH N N NH NH N N ND N NC N P ND N ND P N N P N N ND ND P N N NH N N ND ND N N P N N N N N P NC ND P P ND N P P N N N P N N N P N N NH NH P N
N N N ND ND ND ND P N N ND N ND ND ND ND N P ND N N P N N N ND ND ND ND N N ND NH N N ND N NH ND NH N N N NC ND P N ND P P ND N N P N ND N P N N ND N NH ND P ND P N
Pe P N N N N NC ND NC P P N NC P N N N P ND N N N NH N N N N N NH N P NC NC N N NC NH NC P P N NH N P P P P NC P N ND NH P NH N N N N NH N N NH N N NC N N P
a
d
b
e
N, negative ND, not done c NH, not helpful
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P P N N NH N N P N P ND P N N P P P P N NH P ND NH N P N P N P P ND NC NC N NH N P NC P NC N P N P P NH P P P ND ND NH P ND N P NH ND P N P NH P NH P P NH N
Pyrazinamidase
Reference
M. abscessus M. alvei M. asiaticum M. avium M. bohemicum M. botniense M. branderi M. brumae M. celatum M. chelonae M. chlorophenolicum M. confluentis M. conspicuum M. cookii M. doricum M. elephantis M. flavescens M. fortuitum M. frederiksbergense M. gastri M. genavense M. goodii M. gordonae M. haemophilum M. hassiacum M. heckeshornense M. heidelbergense M. hiberniae M. hodleri M. holsaticum M. immunogenum M. interjectum M. intermedium M. intracellulare M. kansasii M. kubicae M. lacus M. lentiflavum M. madagascariense M. mageritense M. malmoense M. marinum M. montefiorense M. mucogenicum M. murale M. neoaurum M. novocastrense M. palustre M. peregrinum M. phlei M. pinnipedii M. scrofulaceum M. senegalense M. septicum M. shimoidei M. shottsii M. simiae M. smegmatis M. szulgai M. terrae complex M. triplex M. triviale M. tusciae M. ulcerans M. vaccae M. vanbaalenii M. wolinskyi M. xenopi
Urease production
Species
Table 2. Phenotypic characters used for mycobacterial identifications as presented in three current publications (9-11)
P ND N P N ND NC NH P P ND P N ND ND ND P P ND N P ND NH P P N P ND ND P ND NC N P N ND P NH ND P P P ND ND ND P ND P P ND NH NH ND ND P N P ND P NH ND NH NH N P ND NH ND
NC, no consensus P, positive
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© 2006 Elsevier
Clinical Microbiology Newsletter 28:10,2006
There are a number of problems associated with the traditional biochemical identification of mycobacterial species. One of the major problems is related to an increase in the number of new species described over the past few years. Most have been identified by using 16S rRNA nucleic acid sequencing and not by traditional methods, and phenotypic information is not included in the identification tables. These newly described species may have biochemical test results in common with other species, making identification difficult. Most of the data presented in tables have not been updated for years, and the original data have never been verified according to today’s standards. Some reactions are derived from unpublished observations, and some publications fail to list all test reactions, making the list incomplete and unhelpful. In general, traditional biochemical methods require an excessive amount of time before results are available. Mondragon-Barreto et al. (13) showed that the time required for identification of nontuberculous mycobacteria is 30 to 40 days, and Cook et al. (14) support this observation by stating the time required for identification of nontuberculous mycobacteria is between 2 and 8 weeks, depending on the species. Traditional biochemical testing is more costly than some currently used molecular methods such as nucleic acid sequencing. Cook et al. (14) listed the costs as (i) slowly growing mycobacteria that are biochemically active, $80.93; (ii) slowly growing mycobacteria that are biochemically inert, $173.23; and (iii) rapidly growing mycobacteria, $129.40. Reasons for the excessive cost of biochemical testing are related to the time required for periodic reading of tests to determine results, the slowly growing nature of certain species, sometimes inconclusive results of tests that require repeat testing, lack of experience of some laboratory workers in performing tests because of the relative infrequency of their performance, and subjective reading of test results, among others. Tables 3 and 4 show inconsistencies in test results that can cause major problems for even the most experienced persons working in the clinical laboratory. In general, traditional biochemical testing has limited value in the mycoClinical Microbiology Newsletter 28:10,2006
Table 3. Major inconsistencies of biochemical test results as documented in three published references Species
Biochemical test
Ref. 9
Ref. 10
Ref. 11
M. branderi M. celatum M. conspicuum M. doricum M. elephantis M. genavense M. goodii M. kubicae M. kubicae M. mucogenicum M. novocastrense M. shimoidei
Inactivation of catalase at 68˚C Pigment Inactivation of catalase at 68˚C Inactivation of catalase at 68˚C Salt tolerance Tween 80 hydrolysis Inactivation of catalase at 68˚C Nitrate reduction Tween 80 hydrolysis Tellurite reduction Tween 80 hydrolysis Inactivation of catalase at 68˚C
Na N N (100%)d
Pb,c P P Pc P N
N
P N
P N N P N
N N N P P N NHe P N P
a
N, negative P, positive Unpublished d % negative e NH, not helpful; table states positive, but text twice states negative. b c
bacteriology laboratory. It is not as useful as in the past, since it cannot discriminate between the large number of species currently seen in the clinical laboratory and cannot accommodate interspecies variation. Our recommendation is that it not be used at all, except perhaps in instances when a particular test will help, when used in conjunction with another method(s) for identification.
Identification Using HighPerformance Liquid Chromatography High-performance liquid chromatography (HPLC) identification of mycobacteria was proposed in 1985 and introduced into the laboratory during the late 1980s. The principle was based on the analysis of mycolic acids present in the cell wall. The method reduced the time from several weeks to days and became a standard for identification for the CDC (15) in 1990. Funding became available for state health laboratories to upgrade to the latest technology that would allow them to provide accurate and rapid results; this included HPLC. Out of this widely used technology grew a working group that sought to expand its use to identify all species of mycobacteria. In 1996, members assembled a booklet as an initial offering that presented standardized procedures and a collection of chromatographic pattern standards for 23 species of mycobacteria (16). This approach was reported to achieve an accuracy of 96.1% (17). In 1994, the CDC developed a database of chromatograms that contained 45 species; the © 2006 Elsevier
method and library were reported to give an accuracy of greater than 97% (18); however, it is no longer available to users. The Sherlock Mycobacteria Identification System is available from MIDI, Inc. (Newark, DE); the library contains 40 entries that represent 26 species or groups of mycobacteria. It, too, does not yet contain entries for the newly described species. An evaluation of this system by Kellogg et al. (19), using 370 isolates of mycobacteria, showed that 88% were identified to the species level. Overall, it correctly identified 75% of the total number of organisms and 85% of the named species included in the evaluation. The database of chromatograms for the newer species is currently being updated. HPLC is a reliable and accurate method for the identification of several species of mycobacteria. The method is rapid, and results may be available within 2 h; however, instrumentation is costly and trained personnel are necessary to perform the analyses. Unfortunately, no single database of chromatograms is available to all users and any of a number are being used by individual laboratories. The HPLC patterns available for 66 Mycobacterium species may be found in references 15, 20, and 21.
Identification Using Nucleic Acid Probes Even though nucleic acid probe testing is a molecular tool, it is discussed here because it has long been a part of the traditional testing used for the identification of mycobacteria in many 0196-4399/00 (see frontmatter)
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laboratories. Nucleic acid probes used for culture confirmation of mycobacteria became available in the early 1990s and included probes for M. tuberculosis complex, M. avium, M. intracellulare, M. avium-M. intracellulare, M. gordonae, and M. kansasii. Gen-Probe, Inc. (San Diego, CA), developed and marketed these probes (AccuProbes), and they have been of great value when used in combination with colonial morphology and pigmentation characteristics of organisms recovered from clinical specimens. The probes may be used with cultures growing on solid or liquid media, and the accuracy is nearly 100% (22) when growth is sufficient to test. Results are available within 1 h. Unfortunately, there are a limited number of probes available, and it is doubtful whether this will ever change. The development of nucleic acid probes for identification of mycobacteria represents one of the most significant advances in the history of mycobacteriology.
Approach for Identification The routine clinical microbiology laboratory can identify M. avium-M. intracellulare, M. avium, M. intracellulare, M. gordonae, and M. kansasii by using nucleic acid probes, if it is economically feasible to do so. The appropriate probes may be selected by examining cultures to determine whether they are photochromogenic, scotochromogenic, or nonchromogenic by exposing them to the absence and presence of light and observing them for pigment production. The identification of most species of mycobacteria other than M. tuberculosis complex is a difficult task, even for the most experienced laboratory. Fig. 1 presents an algorithm that might be useful to a clinical laboratory that has little expertise in the identification of mycobacteria. It is based on the growth rates of cultures, pigmentation, and use of specific nucleic acid probes. M. aviumM. intracellulare and M. gordonae are some of the most commonly encountered species, and the probes should identify a large proportion of the total organisms recovered from clinical specimens, excluding members of the M. tuberculosis complex. Usually, organisms that do not react with the nucleic acid probes are species less commonly seen, and they require more extensive 78
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measures for identification. Traditionally, biochemical profiles have been used, but with the large number of newly described species now seen, these tests are not useful in today’s laboratory climate where accuracy and rapid turnaround times are so important. It is in the best interest of the laboratory and the patient to refer such isolates to a state health laboratory or to a large reference laboratory that has expertise in mycobacteriology. If laboratories do not perform nucleic acid probe testing, they should refer cultures as described above. The primary physician must always be consulted before cultures are referred to another laboratory to ensure that only those thought to be clinically significant
are sent out for identification. State health laboratories and some reference laboratories use HPLC for identification. This tool is efficient and generally cost-effective, and results are available quickly. 16S rRNA nucleic acid sequencing has taken on a greater role in the identification of mycobacteria. The MicroSeq System (Applied Biosystems, Foster City, CA) is commercially available and has an extensive library of entries for mycobacteria and other bacteria. However, some laboratories have developed custom libraries that accommodate different genotypes and entries for new species as they are described (23,24); these can be used with the MicroSeq system.
Table 4. Minor inconsistencies of biochemical test results as documented in three major published references Species
Biochemical test
Ref. 9
Ref. 10
Ref. 11
M. abscessus M. abscessus M. branderi M. branderi M. brumae M. celatum M. conspicuum M. goodii M. haemophilum M. heidelbergense M. interjectum M. interjectum M. interjectum M. interjectum M. interjectum M. intermedium M. intermedium M. kubicae M. lentiflavum M. lentiflavum M. lentiflavum M. mageritense M. mageritense M. mucogenicum M. novocastrense M. palustre M. shimoidei M. vaccae M. wolinskyi
Salt tolerance Tween 80 hydrolysis Arylsulfatase, 3 days Pyrazinamidase Inactivation of catalase at 68˚C Arylsulfatase, 3 days Arylsulfatase, 3 days Semiquantitative catalase Nitrate reduction Tween 80 hydrolysis Pyrazinamidase Semiquantitative catalase (mm bubbles) Tween 80 hydrolysis Urease production Arylsulfatase, 3 days Urease production Arylsulfatase, 3 days Arylsulfatase, 3 days Semiquantitative catalase (mm bubbles) Arylsulfatase, 3 days Urease production Nitrate reduction Urease production Iron uptake Salt tolerance Arylsulfatase, 3 days Nitrate reduction Arylsulfatase, 3 days Inactivation of catalase at 68˚C
Pa Vc Pe
P/Nb V N P P P Ph 50%i N P P V N P V V Ph
P Nd Pf WPg WP V Ph N V V V V V V WP V Ph V
P
<45e N P P N <45 N P P
V Ph NDj P (70%)i P (60%)i Tank
P N P
N N N/P (100%)n
Nh V V V N WGl V Wm V P
a
P, positive P/N, positive or negative c V, variable d N, negative e Data not published f Positive after 14 days only g WP, weak positive h Positive or negative after 10 days only i % positive j ND, not determined k Neither positive nor negative l WG, weak growth m W, weak n % reaction b
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Clinical Microbiology Newsletter 28:10,2006
Nucleic acid sequencing results may be available within 24 h in some laboratories (24). The major expense associated with HPLC and nucleic acid sequencing is the cost of the equipment. The current emphasis on accuracy and shortened turnaround times makes it easier to justify using these methods. Moreover, as clinical associations and drug resistance profiles are developed for all of the species, these technologies will be essential for good laboratory practice. Conventional methods, except nucleic acid probes, will become extinct, and most clinical laboratories will be unable to offer these methods until costs become affordable. State health laboratories, the CDC, and large reference laboratories will play a greater role in the identification of mycobacteria than in the past, and hospital laboratories will play a lesser role.
Culture: smear positive for acid-fast bacilli
Growth ≥7 days (if known)
Scotochromogenic or photochromogenic (if known)
M. kansasii probe
M. gordonae probe
Growth <7 days (if known)
Nonchromogenic
M. aviumM. intracellulare probe
What About All of These Names? M. tuberculosis is the best-known organism in the genus Mycobacterium, and formerly, it was the only species considered to be important as a cause of human disease. However, it became increasingly apparent that some other mycobacteria recovered from the environment, as well as from clinical specimens, were indeed pathogenic to humans. These additional organisms were called “atypical mycobacteria,” because their appearance was not typical of cultures of M. tuberculosis. Over the years, these mycobacteria have also been referred to as anonymous, unclassified, environmental, opportunistic, nontuberculous mycobacteria, and mycobacteria other than tubercle bacilli. Runyon, in 1965 (25), stated that “use of the terms ‘atypical’ or ‘unclassified’ with respect to the mycobacteria is a matter related not merely to the facilitation of communication: it may involve the propagation of misunderstanding and an impediment to progress. Except to the specialist, the word ‘atypical’ applied to mycobacteria conveys a misconception. It is ill-defined for everyone and tends to emphasize that there is only one mycobacterial pulmonary disease — tuberculosis — and one corresponding pathogen — the tubercle bacillus.” This statement is especially true today, when new species of mycobacteria are being described so frequently. Clinical Microbiology Newsletter 28:10,2006
Probe positive
Probe negative
Biochemical test result
or
Refer to state health or reference laboratory
Figure 1. Algorithm for identification of Mycobacteria (not including M. tuberculosis complex)
Tortoli (2,3) has summarized the diseases caused by 25 of the newly described species, and there are at least 21 additional species known to cause human disease. Many of these mycobacteria exhibit resistance to certain antimicrobials, and the species must be known before appropriate therapy can be initiated. It is important that the clinical microbiologist keep abreast of all new species of mycobacteria and their drug susceptibility profiles, so that he or she can be a resource to the clinician.
The identification of mycobacteria, except in laboratories where nucleic acid probe testing is available, is out of the realm of the routine clinical labora-
tory. Traditional identification methods, such as colonial morphology, pigment production (except when used for the selection of probes), growth rate, optimal growth temperature, and biochemical testing, should be abandoned. All isolates should be sent to a state health laboratory or a commercial reference laboratory so that the organisms can be identified accurately and without delay. Mycobacteria should be referred to by their specific names and should not be lumped into a general category, such as “atypical” or “mycobacteria other than M. tuberculosis.” These categories provide no meaningful information, and it is time for the old terms to be stricken from the vocabulary of the clinician and microbiologist forever.
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References 1. DSMZ. Bacterial nomenclature up-todate. http://www.dsmz.de/bactnom/ bactname.htm. 2. Tortoli, E. 2004. Clinical features of infections caused by new nontuberculous mycobacteria, part I. Clin. Microbiol. Newsl. 26:89-96. 3. Tortoli, E. 2004. Clinical features of infections caused by new nontuberculous mycobacteria, part II. Clin. Microbiol. Newsl. 26:97-104. 4. Debrunner, M. et al. 1992. Epidemiology and clinical significance of nontuberculous mycobacteria in patients negative for human immunodeficiency virus in Switzerland. Clin. Infect. Dis. 15:330-345. 5. Runyon, E.H. 1959. Anonymous mycobacteria in pulmonary disease. Med. Clin. N. Am. 43:273-290. 6. Vestal, A. 1973. Procedures for the isolation and identification of mycobacteria. U.S. Department of Health, Education, and Welfare, Public Health Service, Health Services and Mental Health Administration, Centers for Disease Control, Laboratory Division, Laboratory Consultation and Development Section, Atlanta, GA. 7. Runyon, E.H. 1970. Identification of mycobacterial pathogens utilizing colony characteristics. Am. J. Clin. Pathol. 54:578-586. 8. Welch, D.F. et al. 1993. Timely culture for mycobacteria which utilizes a microcolony method. J. Clin. Microbiol. 31:2178-2184.
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9. Vincent, V. et al. 2003. Mycobacterium: phenotypic and genotypic identification, p. 560-584. In P. R. Murray et al. (ed.), Manual of clinical microbiology, 8th ed. ASM Press. Washington, D.C. 10. Tortoli, E. 2003. Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s. Clin. Microbiol. Rev. 16:319-354. 11. Herdman, A.V. and J.C.H. Steele. 2004. The new mycobacterial species — emerging or newly distinguished pathogens. Clin. Lab. Med. 24:651-690. 12. Witebsky, F.G. and P. Kruczak-Filipov. 1996. Identification of mycobacteria by conventional methods. Clin. Lab. Med. 16:569-601. 13. Mondragon-Barreto, M. et al. 2000. Comparison among three methods for mycobacterial identification. Salud Publica Mex. 42:484-489. 14. Cook, V.J. et al. 2003. Conventional methods versus 16S ribosomal DNA sequencing for identification of nontuberculous mycobacteria: cost analysis. J. Clin. Microbiol. 41:1010-1015. 15. Butler, W.R. and L.S. Guthertz. 2001. Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species. Clin. Microbiol. Rev. 14:704-726. 16. Butler, W.R. et al. 1996. Standardized method for HPLC identification of mycobacteria. Centers for Disease Control and Prevention and U.S. Department of Health and Human Services. Atlanta, GA. 17. Thibert, L. and S. Lapierre. 1993. Routine application of high-performance
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18.
19.
20. 21.
22.
23.
24.
25.
liquid chromatography for identification of mycobacteria. J. Clin. Microbiol. 31:1759-1763. Glickman, S.E. et al. 1994. Rapid identification of mycolic acid patterns of mycobacteria by high-performance liquid chromatography using pattern recognition software and a Mycobacterium library. J. Clin. Microbiol. 32:740-745. Kellogg, J.A. et al. 2001. Application of the Sherlock Mycobacteria Identification System using high-performance liquid chromatography in a clinical laboratory. J. Clin. Microbiol. 39:964-970. The microbiology network user groups. http://microbiol.org/usergroup.htm. MIDI Sherlock Mycobacteria Identification System species list. http://www.midi-inc.com. Woods, G.L. 2002. The mycobacteriology laboratory and new diagnostic techniques. Infect. Dis. Clin. N. Am. 16:127-144. Cloud, J.L. et al. 2002. Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J. Clin. Microbiol. 40:400-406. Hall, L. et al. 2003. Evaluation of the MicroSeq System for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J. Clin. Microbiol. 41:1447-1453. Runyon, E.H. 1965. Typical mycobacteria: their classification. Am. Rev. Respir. Dis. 91:288-289.
Clinical Microbiology Newsletter 28:10,2006
May 15, 2006
Non-Molecular Identification of Nontuberculous Mycobacteria in the Clinical Microbiology Laboratory: What’s the Real Deal? Leslie Hall, M.MSc. and Glenn D. Roberts, Ph.D., Department of Laboratory Medicine and Pathology, Division of Clinical Microbiology, Mayo Clinic and Mayo Clinic College of Medicine, Rochester, Minnesota
Abstract The identification of mycobacteria, other than the Mycobacterium tuberculosis complex, is a challenge for most routine clinical microbiology laboratories. Laboratories may use nucleic acid probe identification of Mycobacterium avium-M. intracellulare, Mycobacterium gordonae, and Mycobacterium kansasii, if financial resources are available. Currently, 109 species of mycobacteria have been described using nucleic acid sequencing. Phenotypic characters are generally incomplete in the literature, and for this reason, it is recommended that routine clinical microbiology laboratories refer cultures to state health laboratories or other reference laboratories for identification.
Introduction In today’s changing world, the clinical microbiology laboratory is faced with numerous challenges, including new technology, expense management, reduced personnel, improving quality, shortened turnaround times, increased costs, and frequent name changes of organisms, among other things. Another challenge of a different type is the identification of mycobacteria. Even though the mycobacteria represent only a small percentage of the organisms seen in the clinical laboratory, they require the greatest amount of time and expertise for recovery and identification. Most routine clinical microbiology laboratories are not experienced enough to offer full mycobacteriology services. The major emphasis in mycobacteriology during recent years has been on the rapid detection and identification of Mycobacterium tuberculosis, due to its
Mailing Address: Dr. Glenn D. Roberts, Division of Clinical Microbiology, Mayo Clinic, Hilton 460C, 200 First Street Southwest, Rochester, MN 55905. Tel.: 507-284-3704. Fax: 507-284-4274. E-mail: [email protected] Clinical Microbiology Newsletter 28:10,2006
public health importance. The Centers for Disease Control and Prevention (CDC) established guidelines for detection and identification of this organism, and every state health laboratory and most laboratories of moderate size are now very familiar with this important pathogen. Molecular methods have enhanced the rapid detection and identification of common mycobacteria, including M. tuberculosis. Unfortunately, smaller clinical microbiology laboratories do not have the necessary resources to accurately identify the mycobacteria and may use a reference laboratory for these services. To complicate matters, there are currently 109 valid species of mycobacteria (1), excluding members of the M. tuberculosis complex and Mycobacterium leprae. Table 1 presents a list of these species and when each was described; many have been associated with human disease, as recently summarized by Tortoliand and Debrunner et al. (2-4). Nucleic acid sequencing was used to identify most of the newly described species, and only a limited number of phenotypic characteristics were published. Interestingly, even © 2006 Elsevier
though each organism has a species name, clinicians and laboratorians often still use the collective term “nontuberculous mycobacteria” to describe the group, while many insist on using the term “atypical mycobacteria.” Just how far should the routine clinical microbiology laboratory go in terms of trying to identify mycobacteria to the species level? Most cannot afford to use currently available molecular methods, perhaps with the exception of nucleic acid probes that have been commercially available for many years. The value of non-molecular testing and recommendations for identification of the species of mycobacteria other than the M. tuberculosis complex will be discussed. In addition, some current thoughts about what these mycobacteria should be named will be included.
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Identification of Mycobacteria Other than M. tuberculosis Complex and M. leprae Identification by colonial morphologic features In 1959, Ernest Runyon (5) developed a classification scheme that placed the common mycobacteria, other than the M. tuberculosis complex, into four groups based on the presence or absence of pigment production: group I, photochromogenic species; group II, scotochromogenic species; group III, nonchromogenic species; and group IV, rapidly growing species. This scheme was helpful to the clinical laboratory when trying to identify the limited number of mycobacteria recovered from clinical specimens at that time. Vestal and Kubica (6), in 1966, described the differential colonial morphologic features of mycobacteria on Middlebrook and Cohn 7H10 agars. Colonies were examined microscopically to determine if they were smooth or rough, pigmented or not, domed or spreading, transparent or not, filamentous or not, and the growth rate was also considered. In 1970, Runyon (7) refined the process and showed that M. tuberculosis, Mycobacterium kansasii, Mycobacterium avium-M. intracellulare, Mycobacterium xenopi, Mycobacterium fortuitum, and Mycobacterium abscessus could be distinguished from each other. He provided illustrative photographs of the colonies of mycobacteria using lowpower microscopy and a limited discussion of the morphologic and biochemical features of all the species; he also included information on Mycobacterium ulcerans, Mycobacterium scrofulaceum, and Mycobacterium marinum. In 1993, Welch et al. (8) reintroduced the microscopic observation of mycobacterial colonies, although this time the emphasis was on the time to
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recovery and identification. These characteristics were helpful for identifying some species, but because the information was not available for many species, some might have had overlapping features. The Runyon grouping provided a useful means of grouping the mycobacteria with similar characteristics, and it is still used in many laboratories today to separate these organisms into these broad categories. However, the groupings are not homogenous, and the distinctions between them are not definitive. Most descriptions are limited to whether a colony is rough or smooth, and this information alone is not helpful, since many of the species share the same features. It should be emphasized that this classification scheme is now largely obsolete due to the large number of species of mycobacteria that have similar morphologic features; it should be used only to broadly define pigmentation characteristics or to describe the growth rate, i.e., rapidly growing species. Pigment production. In general, information related to pigment production of colonies requires that cultures be incubated in the presence and absence of light. Since cultures are not routinely incubated under both conditions, the person who examines the cultures must make a conscious decision as to whether cultures are incubated in the light or dark. If incubators are free from light and doors have been kept closed to prevent exposure of cultures to light, then pigmented colonies may be considered to be scotochromogenic mycobacteria. If organisms are suspected to be photochromogenic, they must be incubated in the presence and absence of light. We reviewed the phenotypic information presented in three recent publications (9-11) and noted that information was available for 68 species (62.4% of described species) of
© 2006 Elsevier
mycobacteria, and of these, only 88.2% described pigment production. Some variation occurs within species; most strains of M. avium-M. intracellulare are nonchromogenic, while others are scotochromogenic. Mycobacterium szulgai is photochromogenic at 25ºC and photochromogenic at 37ºC. As shown in Table 2, there is no consensus on whether Mycobacterium celatum is pigmented. Growth rate The growth rate has been defined by some as the time interval between culturing a clinical specimen and the time that mature colonies are observed. Rapidly growing mycobacteria are said to appear within 7 days, and those that appear later are classified as slowly growing species. There are several inherent problems with this concept. The growth actually depends on a number of factors, including the metabolic rate of an organism, the number of CFU present in the clinical specimen, how often cultures are examined during their incubation, and the medium on which the culture is growing. In reality, a few cells of a rapidly growing species present in a clinical specimen may require more than 7 days for growth, and conversely, a large number of cells of a slowly growing species may require less than 7 days for growth to appear. These are not insignificant problems, and the clinical microbiology laboratory must consider this when using the growth rate as a phenotypic character. Many clinical microbiology laboratories read cultures once weekly, and the exact growth rate cannot be determined. A subculture of an organism that is observed frequently is the best way to determine whether an organism is a rapidly or slowly growing species. However, this adds a significant amount of time to the already slow process of identifying the mycobacteria.
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Acid-fast stain The acid-fast stain is another traditional method that has been used for many years to detect mycobacteria present in clinical specimens; it has also been used to stain cultures to determine whether they have the characteristics of a mycobacterium. The method cannot distinguish one species from another, but it is very important to determine whether an organism is acidfast when trying to place it in the genus Mycobacterium or a related genus.
Table 1. Species of mycobacteria other than members of the M. tuberculosis complex and M. leprae listed by the date they were first described
Optimal growth temperature The optimal growth temperature is a phenotypic character that has been helpful as an aid for the identification of a limited number of mycobacteria. In general, Mycobacterium thermoresistibile grows best at 45ºC, while 42ºC is optimal for M. xenopi. Isolates of M. marinum, Mycobacterium haemophilum, and M. ulcerans prefer temperatures of 30ºC; however, other species share this trait. In general, temperature studies are useful for the separation of only a very limited number of common species.
Identification Using Traditional Biochemical Testing Traditional biochemical tests have been used with some success since the late 1950s and early 1960s to determine the phenotypic characters of some commonly encountered species of mycobacteria. They have been used in combination with colonial morphology, pigment production, and growth rate, along with the acid-fast stain. Representative examples are nitrate reduction, catalase production, catalase inactivation at 68ºC, hydrolysis of Tween, reduction of potassium tellurite, tolerance of 5% NaCl, iron uptake, and arylsulfatase and urease production. A detailed discussion of each test is available in the literature (9,12). Until recently, many of the common species of mycobacteria seen in clinical laboratories were identified using traditional methods. Interestingly, in our experience, biochemical tests failed to provide a definitive identification in many instances, and some were made on a “best-fit” basis when biochemical profiles were considered. Table 2 presents the most common biochemical tests used to aid in the identification of mycobacteria. Clinical Microbiology Newsletter 28:10,2006
Year
Species
Year
Species
1889
M. smegmatis
1987
M. poriferae
1899
M. phlei
1990
1901
M. avium
1912
M. lepraemurium
M. avium spp. paratuberculosis M. avium spp. silvaticum M. cookii
1923
M. chelonae M. paratuberculosis
1992
1926
M. marinum
M. peregrinum M. alvei M. confluentis M. madagascariense
1938
M. fortuitum spp. M. fortuitum
1993
1949
M. intracellulare
1950
M. ulcerans
1953
M. abscessus
M. brumae M. celatum M. genavense M. hiberniae M. intermedium
1955
M. kansasii
1995
1956
M. scrofulaceum
1959
M. xenopi
M. branderi M. interjectum M. mucogenicum
1962
M. flavescens M. gordonae
1996
1964
M. vaccae
M. conspicuum M. lentiflavum M. aurum M. hodleri
1965
M. simiae M. nonchromogenicum M. parafortuitum
1997
1966
M. gastri M. terrae complex M. thermoresistibile
M. hassicum M. mageritense M. triplex M. novocastrense
1998
M. bohemicum M. heidlebergense
1967
M. chitae
1999
1970
M. triviale
1971
M. asiaticum M. duvali M. gilvum M. obuense
M. goodii M. murale M. tusciae M. wolinskyi
2000
M. botniense M. elephantis M. kubicae M. septicum
2001
M. doricum M. fredriksbergense M. heckeshornense M. immunogenum
2002
M. holasaticum M. lacus M. palustre M. vanbaalenii
2003
M. montefiorense M. pinnipedii
2004
M. boenickei M. brisbanense M. canariasense M. chimaera M. cosmeticum M. houstonense M. nebraskense M. neworleansense M. parascrofulaceum M. paramense M. psychrotolerans M. pyrenivorans M. saskatchewanense M. shottsii
1972
M. szulgai M. neoaurum
1973
M. farcinogenes M. senegalense
1974
M. gadium
1977
M. malmoense
1978
M. haemophilum
1979
M. komossense
1980
M. sphagni
1981
M. achiense M. agri M. chubuense M. rhodesiae M. tokaiense
1982
M. shimoidei
1983
M. austroafricanum M. diernhoferi M. fallax M. porcinum M. pulveris
1986
M. fortuitum spp. M. acetamidolyticum M. chlorophenolicum M. moriokaense
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Pigmentation
Niacin production
Carboxylic acid hydrazide
Nitrate reduction
Semiquantitative catalase (mm of bubbles)
Inactivation of catalase at 68˚C
Tween 80 hydrolysis
Tellurite reduction
Salt tolerance
Iron uptake
Arylsulfatase
9-11 10,11 9 9 9-11 10 9-11 10,11 9-11 9 10 10,11 9-11 10 10,11 10,11 9 9 10 9 9-11 9-11 9 9, 11 10,11 9-11 9-11 10 10 10,11 9-11 9-11 9-11 9 9 10,11 10,11 9-11 10 9-11 9 9 11 9-11 10,11 11 10,11 10,11 10,11 9 11 9 9 9-11 9,11 10,11 9 9 9 9 9-11 9 9-11 9 9,11 10 9-11 9
Na N P N P P N N NC N P N P P P P P N P N N NC P N P P N P P N N P P N P P N P P N N P ND N P ND P P N P ND P N N N ND P NH P N N N P N P P N NH
NDb N N N N ND N N N NH ND ND ND ND N ND N N ND N N N N N N N N ND ND ND N N NC N N ND N N ND ND N NH N N ND N N ND ND N NH N N N N P NH N N N N N N N N ND N N
ND ND P P ND ND ND ND P P ND ND P ND P N P P ND P P ND P P NH ND ND ND ND P ND P NH P P ND ND P ND P P P ND ND ND P P ND ND P N P ND ND P P P P P P P P P P P ND ND P
N P N N N N N P N n N P N N P P P P P N N P N NC N N N P N P N N N N P NC P N N NC N N N NH N ND P NH P P N N P P NC N N P P NH P P P N P P P N
>45 >45 >45 <45 <45 <45 <45 >45 <45 >45 ND >45 <45 ND <45 >45 >45 >45 ND <45 >45 NC >45 <45 >45 <45 N <45 ND ND ND NC >45 <45 >45 >45 <45 NC ND NH <45 <45 <45 >45 >45§ <45 <45 <45 >45 >45 ND >45 ND ND <45 <45 >45 <45 >45 >45 >45 >45 <45 <45 >45 ND <45 <45
NHc P P NH P N NC NC P NH ND P NC ND NC P P P ND N P NC P N P P P P ND N ND P P NH P ND N NH ND N NH N N N NH NH P P P P ND P NH ND NC NH P N P P P P P P P ND NC NH
NCd P P N N N N P N NH NH N P N N P P NH P P NC NH P N N N NC ND P NH ND NC P N P NC NH N P N P P N P P P NC P NC P ND N ND ND P N N P NH P N P P N P P NH P
ND ND N P ND ND ND NH P P ND ND N ND P N NH P ND NH N NH N N ND ND N ND ND P NH NH NH P NH ND P N ND NH P NH P NC ND NH ND ND ND P ND NH P ND ND ND P P NH NH ND N NH ND P ND NH NH
NC N N N N ND NH N N NH NH N N ND N NC N P ND N ND P N N P N N ND ND P N N NH N N ND ND N N P N N N N N P NC ND P P ND N P P N N N P N N N P N N NH NH P N
N N N ND ND ND ND P N N ND N ND ND ND ND N P ND N N P N N N ND ND ND ND N N ND NH N N ND N NH ND NH N N N NC ND P N ND P P ND N N P N ND N P N N ND N NH ND P ND P N
Pe P N N N N NC ND NC P P N NC P N N N P ND N N N NH N N N N N NH N P NC NC N N NC NH NC P P N NH N P P P P NC P N ND NH P NH N N N N NH N N NH N N NC N N P
a
d
b
e
N, negative ND, not done c NH, not helpful
76
P P N N NH N N P N P ND P N N P P P P N NH P ND NH N P N P N P P ND NC NC N NH N P NC P NC N P N P P NH P P P ND ND NH P ND N P NH ND P N P NH P NH P P NH N
Pyrazinamidase
Reference
M. abscessus M. alvei M. asiaticum M. avium M. bohemicum M. botniense M. branderi M. brumae M. celatum M. chelonae M. chlorophenolicum M. confluentis M. conspicuum M. cookii M. doricum M. elephantis M. flavescens M. fortuitum M. frederiksbergense M. gastri M. genavense M. goodii M. gordonae M. haemophilum M. hassiacum M. heckeshornense M. heidelbergense M. hiberniae M. hodleri M. holsaticum M. immunogenum M. interjectum M. intermedium M. intracellulare M. kansasii M. kubicae M. lacus M. lentiflavum M. madagascariense M. mageritense M. malmoense M. marinum M. montefiorense M. mucogenicum M. murale M. neoaurum M. novocastrense M. palustre M. peregrinum M. phlei M. pinnipedii M. scrofulaceum M. senegalense M. septicum M. shimoidei M. shottsii M. simiae M. smegmatis M. szulgai M. terrae complex M. triplex M. triviale M. tusciae M. ulcerans M. vaccae M. vanbaalenii M. wolinskyi M. xenopi
Urease production
Species
Table 2. Phenotypic characters used for mycobacterial identifications as presented in three current publications (9-11)
P ND N P N ND NC NH P P ND P N ND ND ND P P ND N P ND NH P P N P ND ND P ND NC N P N ND P NH ND P P P ND ND ND P ND P P ND NH NH ND ND P N P ND P NH ND NH NH N P ND NH ND
NC, no consensus P, positive
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Clinical Microbiology Newsletter 28:10,2006
There are a number of problems associated with the traditional biochemical identification of mycobacterial species. One of the major problems is related to an increase in the number of new species described over the past few years. Most have been identified by using 16S rRNA nucleic acid sequencing and not by traditional methods, and phenotypic information is not included in the identification tables. These newly described species may have biochemical test results in common with other species, making identification difficult. Most of the data presented in tables have not been updated for years, and the original data have never been verified according to today’s standards. Some reactions are derived from unpublished observations, and some publications fail to list all test reactions, making the list incomplete and unhelpful. In general, traditional biochemical methods require an excessive amount of time before results are available. Mondragon-Barreto et al. (13) showed that the time required for identification of nontuberculous mycobacteria is 30 to 40 days, and Cook et al. (14) support this observation by stating the time required for identification of nontuberculous mycobacteria is between 2 and 8 weeks, depending on the species. Traditional biochemical testing is more costly than some currently used molecular methods such as nucleic acid sequencing. Cook et al. (14) listed the costs as (i) slowly growing mycobacteria that are biochemically active, $80.93; (ii) slowly growing mycobacteria that are biochemically inert, $173.23; and (iii) rapidly growing mycobacteria, $129.40. Reasons for the excessive cost of biochemical testing are related to the time required for periodic reading of tests to determine results, the slowly growing nature of certain species, sometimes inconclusive results of tests that require repeat testing, lack of experience of some laboratory workers in performing tests because of the relative infrequency of their performance, and subjective reading of test results, among others. Tables 3 and 4 show inconsistencies in test results that can cause major problems for even the most experienced persons working in the clinical laboratory. In general, traditional biochemical testing has limited value in the mycoClinical Microbiology Newsletter 28:10,2006
Table 3. Major inconsistencies of biochemical test results as documented in three published references Species
Biochemical test
Ref. 9
Ref. 10
Ref. 11
M. branderi M. celatum M. conspicuum M. doricum M. elephantis M. genavense M. goodii M. kubicae M. kubicae M. mucogenicum M. novocastrense M. shimoidei
Inactivation of catalase at 68˚C Pigment Inactivation of catalase at 68˚C Inactivation of catalase at 68˚C Salt tolerance Tween 80 hydrolysis Inactivation of catalase at 68˚C Nitrate reduction Tween 80 hydrolysis Tellurite reduction Tween 80 hydrolysis Inactivation of catalase at 68˚C
Na N N (100%)d
Pb,c P P Pc P N
N
P N
P N N P N
N N N P P N NHe P N P
a
N, negative P, positive Unpublished d % negative e NH, not helpful; table states positive, but text twice states negative. b c
bacteriology laboratory. It is not as useful as in the past, since it cannot discriminate between the large number of species currently seen in the clinical laboratory and cannot accommodate interspecies variation. Our recommendation is that it not be used at all, except perhaps in instances when a particular test will help, when used in conjunction with another method(s) for identification.
Identification Using HighPerformance Liquid Chromatography High-performance liquid chromatography (HPLC) identification of mycobacteria was proposed in 1985 and introduced into the laboratory during the late 1980s. The principle was based on the analysis of mycolic acids present in the cell wall. The method reduced the time from several weeks to days and became a standard for identification for the CDC (15) in 1990. Funding became available for state health laboratories to upgrade to the latest technology that would allow them to provide accurate and rapid results; this included HPLC. Out of this widely used technology grew a working group that sought to expand its use to identify all species of mycobacteria. In 1996, members assembled a booklet as an initial offering that presented standardized procedures and a collection of chromatographic pattern standards for 23 species of mycobacteria (16). This approach was reported to achieve an accuracy of 96.1% (17). In 1994, the CDC developed a database of chromatograms that contained 45 species; the © 2006 Elsevier
method and library were reported to give an accuracy of greater than 97% (18); however, it is no longer available to users. The Sherlock Mycobacteria Identification System is available from MIDI, Inc. (Newark, DE); the library contains 40 entries that represent 26 species or groups of mycobacteria. It, too, does not yet contain entries for the newly described species. An evaluation of this system by Kellogg et al. (19), using 370 isolates of mycobacteria, showed that 88% were identified to the species level. Overall, it correctly identified 75% of the total number of organisms and 85% of the named species included in the evaluation. The database of chromatograms for the newer species is currently being updated. HPLC is a reliable and accurate method for the identification of several species of mycobacteria. The method is rapid, and results may be available within 2 h; however, instrumentation is costly and trained personnel are necessary to perform the analyses. Unfortunately, no single database of chromatograms is available to all users and any of a number are being used by individual laboratories. The HPLC patterns available for 66 Mycobacterium species may be found in references 15, 20, and 21.
Identification Using Nucleic Acid Probes Even though nucleic acid probe testing is a molecular tool, it is discussed here because it has long been a part of the traditional testing used for the identification of mycobacteria in many 0196-4399/00 (see frontmatter)
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laboratories. Nucleic acid probes used for culture confirmation of mycobacteria became available in the early 1990s and included probes for M. tuberculosis complex, M. avium, M. intracellulare, M. avium-M. intracellulare, M. gordonae, and M. kansasii. Gen-Probe, Inc. (San Diego, CA), developed and marketed these probes (AccuProbes), and they have been of great value when used in combination with colonial morphology and pigmentation characteristics of organisms recovered from clinical specimens. The probes may be used with cultures growing on solid or liquid media, and the accuracy is nearly 100% (22) when growth is sufficient to test. Results are available within 1 h. Unfortunately, there are a limited number of probes available, and it is doubtful whether this will ever change. The development of nucleic acid probes for identification of mycobacteria represents one of the most significant advances in the history of mycobacteriology.
Approach for Identification The routine clinical microbiology laboratory can identify M. avium-M. intracellulare, M. avium, M. intracellulare, M. gordonae, and M. kansasii by using nucleic acid probes, if it is economically feasible to do so. The appropriate probes may be selected by examining cultures to determine whether they are photochromogenic, scotochromogenic, or nonchromogenic by exposing them to the absence and presence of light and observing them for pigment production. The identification of most species of mycobacteria other than M. tuberculosis complex is a difficult task, even for the most experienced laboratory. Fig. 1 presents an algorithm that might be useful to a clinical laboratory that has little expertise in the identification of mycobacteria. It is based on the growth rates of cultures, pigmentation, and use of specific nucleic acid probes. M. aviumM. intracellulare and M. gordonae are some of the most commonly encountered species, and the probes should identify a large proportion of the total organisms recovered from clinical specimens, excluding members of the M. tuberculosis complex. Usually, organisms that do not react with the nucleic acid probes are species less commonly seen, and they require more extensive 78
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measures for identification. Traditionally, biochemical profiles have been used, but with the large number of newly described species now seen, these tests are not useful in today’s laboratory climate where accuracy and rapid turnaround times are so important. It is in the best interest of the laboratory and the patient to refer such isolates to a state health laboratory or to a large reference laboratory that has expertise in mycobacteriology. If laboratories do not perform nucleic acid probe testing, they should refer cultures as described above. The primary physician must always be consulted before cultures are referred to another laboratory to ensure that only those thought to be clinically significant
are sent out for identification. State health laboratories and some reference laboratories use HPLC for identification. This tool is efficient and generally cost-effective, and results are available quickly. 16S rRNA nucleic acid sequencing has taken on a greater role in the identification of mycobacteria. The MicroSeq System (Applied Biosystems, Foster City, CA) is commercially available and has an extensive library of entries for mycobacteria and other bacteria. However, some laboratories have developed custom libraries that accommodate different genotypes and entries for new species as they are described (23,24); these can be used with the MicroSeq system.
Table 4. Minor inconsistencies of biochemical test results as documented in three major published references Species
Biochemical test
Ref. 9
Ref. 10
Ref. 11
M. abscessus M. abscessus M. branderi M. branderi M. brumae M. celatum M. conspicuum M. goodii M. haemophilum M. heidelbergense M. interjectum M. interjectum M. interjectum M. interjectum M. interjectum M. intermedium M. intermedium M. kubicae M. lentiflavum M. lentiflavum M. lentiflavum M. mageritense M. mageritense M. mucogenicum M. novocastrense M. palustre M. shimoidei M. vaccae M. wolinskyi
Salt tolerance Tween 80 hydrolysis Arylsulfatase, 3 days Pyrazinamidase Inactivation of catalase at 68˚C Arylsulfatase, 3 days Arylsulfatase, 3 days Semiquantitative catalase Nitrate reduction Tween 80 hydrolysis Pyrazinamidase Semiquantitative catalase (mm bubbles) Tween 80 hydrolysis Urease production Arylsulfatase, 3 days Urease production Arylsulfatase, 3 days Arylsulfatase, 3 days Semiquantitative catalase (mm bubbles) Arylsulfatase, 3 days Urease production Nitrate reduction Urease production Iron uptake Salt tolerance Arylsulfatase, 3 days Nitrate reduction Arylsulfatase, 3 days Inactivation of catalase at 68˚C
Pa Vc Pe
P/Nb V N P P P Ph 50%i N P P V N P V V Ph
P Nd Pf WPg WP V Ph N V V V V V V WP V Ph V
P
<45e N P P N <45 N P P
V Ph NDj P (70%)i P (60%)i Tank
P N P
N N N/P (100%)n
Nh V V V N WGl V Wm V P
a
P, positive P/N, positive or negative c V, variable d N, negative e Data not published f Positive after 14 days only g WP, weak positive h Positive or negative after 10 days only i % positive j ND, not determined k Neither positive nor negative l WG, weak growth m W, weak n % reaction b
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Nucleic acid sequencing results may be available within 24 h in some laboratories (24). The major expense associated with HPLC and nucleic acid sequencing is the cost of the equipment. The current emphasis on accuracy and shortened turnaround times makes it easier to justify using these methods. Moreover, as clinical associations and drug resistance profiles are developed for all of the species, these technologies will be essential for good laboratory practice. Conventional methods, except nucleic acid probes, will become extinct, and most clinical laboratories will be unable to offer these methods until costs become affordable. State health laboratories, the CDC, and large reference laboratories will play a greater role in the identification of mycobacteria than in the past, and hospital laboratories will play a lesser role.
Culture: smear positive for acid-fast bacilli
Growth ≥7 days (if known)
Scotochromogenic or photochromogenic (if known)
M. kansasii probe
M. gordonae probe
Growth <7 days (if known)
Nonchromogenic
M. aviumM. intracellulare probe
What About All of These Names? M. tuberculosis is the best-known organism in the genus Mycobacterium, and formerly, it was the only species considered to be important as a cause of human disease. However, it became increasingly apparent that some other mycobacteria recovered from the environment, as well as from clinical specimens, were indeed pathogenic to humans. These additional organisms were called “atypical mycobacteria,” because their appearance was not typical of cultures of M. tuberculosis. Over the years, these mycobacteria have also been referred to as anonymous, unclassified, environmental, opportunistic, nontuberculous mycobacteria, and mycobacteria other than tubercle bacilli. Runyon, in 1965 (25), stated that “use of the terms ‘atypical’ or ‘unclassified’ with respect to the mycobacteria is a matter related not merely to the facilitation of communication: it may involve the propagation of misunderstanding and an impediment to progress. Except to the specialist, the word ‘atypical’ applied to mycobacteria conveys a misconception. It is ill-defined for everyone and tends to emphasize that there is only one mycobacterial pulmonary disease — tuberculosis — and one corresponding pathogen — the tubercle bacillus.” This statement is especially true today, when new species of mycobacteria are being described so frequently. Clinical Microbiology Newsletter 28:10,2006
Probe positive
Probe negative
Biochemical test result
or
Refer to state health or reference laboratory
Figure 1. Algorithm for identification of Mycobacteria (not including M. tuberculosis complex)
Tortoli (2,3) has summarized the diseases caused by 25 of the newly described species, and there are at least 21 additional species known to cause human disease. Many of these mycobacteria exhibit resistance to certain antimicrobials, and the species must be known before appropriate therapy can be initiated. It is important that the clinical microbiologist keep abreast of all new species of mycobacteria and their drug susceptibility profiles, so that he or she can be a resource to the clinician.
The identification of mycobacteria, except in laboratories where nucleic acid probe testing is available, is out of the realm of the routine clinical labora-
tory. Traditional identification methods, such as colonial morphology, pigment production (except when used for the selection of probes), growth rate, optimal growth temperature, and biochemical testing, should be abandoned. All isolates should be sent to a state health laboratory or a commercial reference laboratory so that the organisms can be identified accurately and without delay. Mycobacteria should be referred to by their specific names and should not be lumped into a general category, such as “atypical” or “mycobacteria other than M. tuberculosis.” These categories provide no meaningful information, and it is time for the old terms to be stricken from the vocabulary of the clinician and microbiologist forever.
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The Real Deal
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References 1. DSMZ. Bacterial nomenclature up-todate. http://www.dsmz.de/bactnom/ bactname.htm. 2. Tortoli, E. 2004. Clinical features of infections caused by new nontuberculous mycobacteria, part I. Clin. Microbiol. Newsl. 26:89-96. 3. Tortoli, E. 2004. Clinical features of infections caused by new nontuberculous mycobacteria, part II. Clin. Microbiol. Newsl. 26:97-104. 4. Debrunner, M. et al. 1992. Epidemiology and clinical significance of nontuberculous mycobacteria in patients negative for human immunodeficiency virus in Switzerland. Clin. Infect. Dis. 15:330-345. 5. Runyon, E.H. 1959. Anonymous mycobacteria in pulmonary disease. Med. Clin. N. Am. 43:273-290. 6. Vestal, A. 1973. Procedures for the isolation and identification of mycobacteria. U.S. Department of Health, Education, and Welfare, Public Health Service, Health Services and Mental Health Administration, Centers for Disease Control, Laboratory Division, Laboratory Consultation and Development Section, Atlanta, GA. 7. Runyon, E.H. 1970. Identification of mycobacterial pathogens utilizing colony characteristics. Am. J. Clin. Pathol. 54:578-586. 8. Welch, D.F. et al. 1993. Timely culture for mycobacteria which utilizes a microcolony method. J. Clin. Microbiol. 31:2178-2184.
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9. Vincent, V. et al. 2003. Mycobacterium: phenotypic and genotypic identification, p. 560-584. In P. R. Murray et al. (ed.), Manual of clinical microbiology, 8th ed. ASM Press. Washington, D.C. 10. Tortoli, E. 2003. Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s. Clin. Microbiol. Rev. 16:319-354. 11. Herdman, A.V. and J.C.H. Steele. 2004. The new mycobacterial species — emerging or newly distinguished pathogens. Clin. Lab. Med. 24:651-690. 12. Witebsky, F.G. and P. Kruczak-Filipov. 1996. Identification of mycobacteria by conventional methods. Clin. Lab. Med. 16:569-601. 13. Mondragon-Barreto, M. et al. 2000. Comparison among three methods for mycobacterial identification. Salud Publica Mex. 42:484-489. 14. Cook, V.J. et al. 2003. Conventional methods versus 16S ribosomal DNA sequencing for identification of nontuberculous mycobacteria: cost analysis. J. Clin. Microbiol. 41:1010-1015. 15. Butler, W.R. and L.S. Guthertz. 2001. Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species. Clin. Microbiol. Rev. 14:704-726. 16. Butler, W.R. et al. 1996. Standardized method for HPLC identification of mycobacteria. Centers for Disease Control and Prevention and U.S. Department of Health and Human Services. Atlanta, GA. 17. Thibert, L. and S. Lapierre. 1993. Routine application of high-performance
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liquid chromatography for identification of mycobacteria. J. Clin. Microbiol. 31:1759-1763. Glickman, S.E. et al. 1994. Rapid identification of mycolic acid patterns of mycobacteria by high-performance liquid chromatography using pattern recognition software and a Mycobacterium library. J. Clin. Microbiol. 32:740-745. Kellogg, J.A. et al. 2001. Application of the Sherlock Mycobacteria Identification System using high-performance liquid chromatography in a clinical laboratory. J. Clin. Microbiol. 39:964-970. The microbiology network user groups. http://microbiol.org/usergroup.htm. MIDI Sherlock Mycobacteria Identification System species list. http://www.midi-inc.com. Woods, G.L. 2002. The mycobacteriology laboratory and new diagnostic techniques. Infect. Dis. Clin. N. Am. 16:127-144. Cloud, J.L. et al. 2002. Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries. J. Clin. Microbiol. 40:400-406. Hall, L. et al. 2003. Evaluation of the MicroSeq System for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J. Clin. Microbiol. 41:1447-1453. Runyon, E.H. 1965. Typical mycobacteria: their classification. Am. Rev. Respir. Dis. 91:288-289.
Clinical Microbiology Newsletter 28:10,2006