Anthrax other diagnostic studies: Difference between revisions
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{{Anthrax}} | {{Anthrax}} | ||
== | ==Overview== | ||
Various techniques are used for the direct identification of ''B. anthracis'' in clinical material. Firstly, specimens may be [[Gram stain]]ed. ''Bacillus'' spp. are quite large in size (3 to 4 μm long), they grow in long chains, and they stain Gram-positive. To confirm the organism is ''B. anthracis'', rapid diagnostic techniques such as [[polymerase chain reaction]]-based assays and [[Immunofluorescence|immunofluorescence microscopy]] may be used.<ref>{{cite book |author=Levinson, W. |title=Review of Medical Microbiology and Immunology |year=2010 |edition=11th}}</ref> | Various techniques are used for the direct identification of ''B. anthracis'' in clinical material. Firstly, specimens may be [[Gram stain]]ed. ''Bacillus'' spp. are quite large in size (3 to 4 μm long), they grow in long chains, and they stain Gram-positive. To confirm the organism is ''B. anthracis'', rapid diagnostic techniques such as [[polymerase chain reaction]]-based assays and [[Immunofluorescence|immunofluorescence microscopy]] may be used.<ref>{{cite book |author=Levinson, W. |title=Review of Medical Microbiology and Immunology |year=2010 |edition=11th}}</ref> | ||
All ''Bacillus'' species grow well on 5% sheep blood agar and other routine culture media. Polymyxin-lysozyme-EDTA-thallous acetate can be used to isolate ''B. anthracis'' from contaminated specimens, and bicarbonate agar is used as an identification method to induce capsule formation. ''Bacillus'' spp. usually grow within 24 hours of incubation at 35°C, in ambient air (room temperature) or in 5% CO<sub>2</sub>. If bicarbonate agar is used for identification, then the medium must be incubated in 5% CO<sub>2</sub>. ''B. anthracis'' colonies are medium-large, gray, flat, and irregular with swirling projections, often referred to as having a "[[Medusa|medusa head]]" appearance, and are not hemolytic on 5% sheep blood agar. The bacteria are not motile, susceptible to penicillin, and produce a wide zone of lecithinase on egg yolk agar. Confirmatory testing to identify ''B. anthracis'' includes gamma bacteriophage testing, indirect hemagglutination, and enzyme linked immunosorbent assay to detect antibodies.<ref>{{cite book |author=Forbes, B.A. |title=Bailey & Scott's Diagnostic Microbiology |year=2002 |edition=11th}}</ref> The best confirmatory precipitation test for anthrax is the [[Alberto Ascoli|Ascoli]] test. | All ''Bacillus'' species grow well on 5% sheep blood agar and other routine culture media. Polymyxin-lysozyme-EDTA-thallous acetate can be used to isolate ''B. anthracis'' from contaminated specimens, and bicarbonate agar is used as an identification method to induce capsule formation. ''Bacillus'' spp. usually grow within 24 hours of incubation at 35°C, in ambient air (room temperature) or in 5% CO<sub>2</sub>. If bicarbonate agar is used for identification, then the medium must be incubated in 5% CO<sub>2</sub>. ''B. anthracis'' colonies are medium-large, gray, flat, and irregular with swirling projections, often referred to as having a "[[Medusa|medusa head]]" appearance, and are not hemolytic on 5% sheep blood agar. The bacteria are not motile, susceptible to penicillin, and produce a wide zone of lecithinase on egg yolk agar. Confirmatory testing to identify ''B. anthracis'' includes gamma bacteriophage testing, indirect hemagglutination, and enzyme linked immunosorbent assay to detect antibodies.<ref>{{cite book |author=Forbes, B.A. |title=Bailey & Scott's Diagnostic Microbiology |year=2002 |edition=11th}}</ref> The best confirmatory precipitation test for anthrax is the [[Alberto Ascoli|Ascoli]] test. | ||
==Diagnostic Studies== | |||
===Hemolysis=== | ===Hemolysis=== | ||
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===Motility=== | ===Motility=== | ||
Although examination for motility is always listed as one of the primary identification tests for B. anthracis, it is doubtful that the test is often done on new isolates or checked more than rudimentarily and occasionally on culture collection cultures. Certainly, the first obvious appearance for diagnostic test purposes is lack of motility, but in view of the existence of genes associated with motility, and even one recent report that includes electron micrographs showing flagella, perhaps the “absoluteness” of non-motility in B. anthracis should be revisited. | Although examination for motility is always listed as one of the primary identification tests for B. anthracis, it is doubtful that the test is often done on new isolates or checked more than rudimentarily and occasionally on culture collection cultures. Certainly, the first obvious appearance for diagnostic test purposes is lack of motility, but in view of the existence of genes associated with motility, and even one recent report that includes electron micrographs showing flagella, perhaps the “absoluteness” of non-motility in B. anthracis should be revisited. | ||
===Diagnostic phage=== | |||
Just to what extent the diagnostic phages used in various laboratories around the world and often referred to as “gamma phages” are unadulterated descendants of McCloy’s gamma phage is now uncer- tain. For this reason, the term “diagnostic phage” is preferred. | |||
Some care needs to be taken in defining precisely what is meant by phage sensitivity. the titre of the phage suspension is of consequence, and interpreta- tion of what is seen in the zone of effect is somewhat subjective. For the purposes of Table 3, the titre of the phage suspension is ≥ 109 pfu/ml and any zone of effect, which may be graded from ± to 4+, rep- resents sensitivity. (note: this is different from the manner in which antibiotic sensitivity is read, where colonies growing in a zone of clearing result in an interpretation of “resistant”.) For phage sensitivity tests, a total absence of effect is regarded as true resistance. on this basis, in formal studies on sus- ceptibility/resistance, Buck et al. (1963) found that 7 of 264 (2.7%) B. anthracis isolates were phage-resist- ant, and 3 of 64 (3.1%) non-anthrax Bacillus species were lysed by anthrax phage. Similarly, redmond et al. (1996b) found that 1 of 87 (1.2%) strains of B. anthracis were phage-resistant, and 2 of 47 (4.2%) non-anthrax Bacillus species were lysed by anthrax phage. in further tests (unpublished), turnbull and colleagues found no positives among 14 B. cereus, 10 B. megaterium, 6 B. pumilus, 5 B. subtilis, 5 B. circulans, 4 B. mycoides, 4 B. firmus, 4 B. sphaericus, 3 B. licheniformis, 3 B. thuringiensis, 2 B. amyloliquefaciens, 1 B. lentus, and 2 Brevibacillus brevis from collections at the Food Safety and Microbiology Laboratory, health Protection | |||
==PCR== | ==PCR== | ||
PCR is becoming more widely available as a means of confirming the presence of the virulence factor (capsule and toxin) genes, and hence that an isolate is, or is not, virulent B. anthracis. For routine purposes, primers to one of the toxin genes (usually the Protective Antigen gene) and to one of the enzymes mediating capsule formation are adequate. In laboratories not equipped for PCR tests, if doubt remains to the definitive identity of a suspect B. anthracis isolate, inoculation into a mouse or guinea-pig may be the only way remaining to determine conclusively if it is virulent B. anthracis. However this should be a last resort procedure and confined to situations where a definitive identification is essential. | PCR is becoming more widely available as a means of confirming the presence of the virulence factor (capsule and toxin) genes, and hence that an isolate is, or is not, virulent B. anthracis. For routine purposes, primers to one of the toxin genes (usually the Protective Antigen gene) and to one of the enzymes mediating capsule formation are adequate. In laboratories not equipped for PCR tests, if doubt remains to the definitive identity of a suspect B. anthracis isolate, inoculation into a mouse or guinea-pig may be the only way remaining to determine conclusively if it is virulent B. anthracis. However this should be a last resort procedure and confined to situations where a definitive identification is essential. | ||
==References== | ==References== | ||
Revision as of 18:42, 16 July 2014
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Overview
Various techniques are used for the direct identification of B. anthracis in clinical material. Firstly, specimens may be Gram stained. Bacillus spp. are quite large in size (3 to 4 μm long), they grow in long chains, and they stain Gram-positive. To confirm the organism is B. anthracis, rapid diagnostic techniques such as polymerase chain reaction-based assays and immunofluorescence microscopy may be used.[1]
All Bacillus species grow well on 5% sheep blood agar and other routine culture media. Polymyxin-lysozyme-EDTA-thallous acetate can be used to isolate B. anthracis from contaminated specimens, and bicarbonate agar is used as an identification method to induce capsule formation. Bacillus spp. usually grow within 24 hours of incubation at 35°C, in ambient air (room temperature) or in 5% CO2. If bicarbonate agar is used for identification, then the medium must be incubated in 5% CO2. B. anthracis colonies are medium-large, gray, flat, and irregular with swirling projections, often referred to as having a "medusa head" appearance, and are not hemolytic on 5% sheep blood agar. The bacteria are not motile, susceptible to penicillin, and produce a wide zone of lecithinase on egg yolk agar. Confirmatory testing to identify B. anthracis includes gamma bacteriophage testing, indirect hemagglutination, and enzyme linked immunosorbent assay to detect antibodies.[2] The best confirmatory precipitation test for anthrax is the Ascoli test.
Diagnostic Studies
Hemolysis
B. anthracis was shown to be haemolytic on blood agar made with sheep red cells that had been washed with buffered saline containing calcium and magnesium. Similarly, the anthrolysin is presumably haemolytic. Reports are also occasionally encountered of haemolysis in blood or on agar containing blood of certain species, including human.
Lecithinase
B. anthracis either produces lecithinase to a lesser extent than its close relatives, B. cereus and B. thuringiensis, or produces a lecithinase with a lower activity. In the lecithinase test on egg yolk agar, the zone of opalescence or “halo” almost always seen around colonies or areas of growth of B. cereus and B. thuringiensis is only sometimes visible around B. anthracis, probably only becoming apparent at 48 hours (35-37 °C) and usually in a narrow band when present. Opalescence should be looked for under the colony/area of growth by scraping away some of the colony material. It can be seen here after 24 hours incubation at 35–37 °C. While B. anthracis grows well on conventional egg yolk agar, it grows less well than B. cereus on Kendall’s BC egg yolk-mannitol agar; the growth of B. anthracis on this medium (24–48 hours) is greyish as compared to the deep purple of B. cereus and, in contrast to B. cereus, a zone of opalescence does not form around the growth of B. anthracis. once again, colony material must be scraped away to see the underlying LV (lecithovitellin) reaction.
Motility
Although examination for motility is always listed as one of the primary identification tests for B. anthracis, it is doubtful that the test is often done on new isolates or checked more than rudimentarily and occasionally on culture collection cultures. Certainly, the first obvious appearance for diagnostic test purposes is lack of motility, but in view of the existence of genes associated with motility, and even one recent report that includes electron micrographs showing flagella, perhaps the “absoluteness” of non-motility in B. anthracis should be revisited.
Diagnostic phage
Just to what extent the diagnostic phages used in various laboratories around the world and often referred to as “gamma phages” are unadulterated descendants of McCloy’s gamma phage is now uncer- tain. For this reason, the term “diagnostic phage” is preferred.
Some care needs to be taken in defining precisely what is meant by phage sensitivity. the titre of the phage suspension is of consequence, and interpreta- tion of what is seen in the zone of effect is somewhat subjective. For the purposes of Table 3, the titre of the phage suspension is ≥ 109 pfu/ml and any zone of effect, which may be graded from ± to 4+, rep- resents sensitivity. (note: this is different from the manner in which antibiotic sensitivity is read, where colonies growing in a zone of clearing result in an interpretation of “resistant”.) For phage sensitivity tests, a total absence of effect is regarded as true resistance. on this basis, in formal studies on sus- ceptibility/resistance, Buck et al. (1963) found that 7 of 264 (2.7%) B. anthracis isolates were phage-resist- ant, and 3 of 64 (3.1%) non-anthrax Bacillus species were lysed by anthrax phage. Similarly, redmond et al. (1996b) found that 1 of 87 (1.2%) strains of B. anthracis were phage-resistant, and 2 of 47 (4.2%) non-anthrax Bacillus species were lysed by anthrax phage. in further tests (unpublished), turnbull and colleagues found no positives among 14 B. cereus, 10 B. megaterium, 6 B. pumilus, 5 B. subtilis, 5 B. circulans, 4 B. mycoides, 4 B. firmus, 4 B. sphaericus, 3 B. licheniformis, 3 B. thuringiensis, 2 B. amyloliquefaciens, 1 B. lentus, and 2 Brevibacillus brevis from collections at the Food Safety and Microbiology Laboratory, health Protection
PCR
PCR is becoming more widely available as a means of confirming the presence of the virulence factor (capsule and toxin) genes, and hence that an isolate is, or is not, virulent B. anthracis. For routine purposes, primers to one of the toxin genes (usually the Protective Antigen gene) and to one of the enzymes mediating capsule formation are adequate. In laboratories not equipped for PCR tests, if doubt remains to the definitive identity of a suspect B. anthracis isolate, inoculation into a mouse or guinea-pig may be the only way remaining to determine conclusively if it is virulent B. anthracis. However this should be a last resort procedure and confined to situations where a definitive identification is essential.