Challenge G094 CMPT Clinical Bacteriology Program

CMPT Clinical Bacteriology Program
Innovation, Education, Quality Assessment, Continual Improvement
Challenge G094
February 2010
Gram: skin aspirate- gram positive bacilli (Clostridium tertium)
HISTORY
The sample was sent to participant laboratories
as a gram smear from a skin aspirate from a 57
year old male, febrile with leukemia, neutropenia
and a blood blister.
CMPT QA
CMPT review indicated 2+ (1-5/oif) neutrophils,
4+ (>10/oif) RBC and 4+ (>50/oif) gram positive
bacilli (see Figure 1). The challenge was
verified by internal quality control, which indicated
99% confidence based on MIL-STD-105E 9
(see Figure 1).
SURVEY RESULTS
Cell component (see table 1) 9/15 labs reported
1+/2+/3+/4+, >25/oif neutrophils (4
added 4+/>10/oif RBCs); 4 labs reported no
neutrophils seen (1 added 4+ RBCs); 1 lab reported
3+ RBCs only and 1 lab reported 1+
neutrophils, 1+ epithelial cells, 4+ RBCs. The
cell component of this challenge was ungraded
because of lack of consensus amongst the reference
labs. This is not surprising since the
sample was prepared to have few neutrophils
because the patient was neutropenic.
Approximately 89% of the laboratories reported
neutrophils in the sample. Of those participants
reporting neutrophils, 31% reported the presence
of RBCs as well, 7% reported the presence
of epithelial cells and 3% reported the presence
of both. Four percent of labs reported only
RBCs and 8 participants reported no cells.
Figure1: G094 gram smear. The smear shows 2+ neutrophils
and 4+ gram positive bacilli. Decolorized bacilli
look like gram negative bacilli.
Bacterial component (see table 2) 15/15 reference
labs reported 3+/4+, >25/oif gram positive
bacilli. Consensus was achieved by the
reference labs thus the sample was suitable for
grading.
The description of the bacterial component of
this challenge was quite varied. Those laboratories
reporting gram positive bacilli (78%), gram
positive bacilli resembling anaerobic bacilli/
Clostridium species (6%), or mix of gram positive
and gram negative bacilli or gram variable
bacilli (14%) were given a grade of 4.
Participants were downgraded to 3 if Bacillus
species was suggested, with no mention of
probable anaerobe involved. Reporting diphtheroids
was considered unacceptable (see
comment on the side) and was graded 1, as
was the reporting of gram negative bacilli only
or gram positive cocci with gram positive or
Table –1: G094 smear reported results - Cell component -
Reported results Total % Grade
1+/2+/3+/4+ Neutrophils and… 117 89
no other cells reported 69 52
>10, >100/oif, 4+RBC 36 27
1+/2+/3+/4+epithelial cells 8 6
1+/2+ epithelial cells + 4+ RBC 4 3 ungraded
3+/4+ RBC's +/- no neutrophils seen 5 4

negative bacilli.
COMMENTS ON RESULTS
Identification of C. tertium can be difficult because
it is often variably stained in gram
smears and can therefore be mistaken as a
gram-negative bacilli 2 . The organisms are
large and may have squared ends. Terminal
spores are produced under anaerobic conditions
4 .
The isolates are often initially considered gramnegative
after overnight incubation, but will
easily be recognized as gram- positive when the
stain is made from cultures grown anaerobically
2,3,4. The aerotolerance of C. tertium, together
with its variable gram staining properties, results
in the potential for misidentification (as a
Bacillus or Corynebacterium species. 5
CLINICAL SIGNIFICANCE
Recognition of the presence of aerotolerant C.
tertium in clinical specimens is important in the
determination of appropriate treatment.
An important concern for microbiologists should
be that an isolate of C. tertium is not prematurely
dismissed as a diphtheroid or a probable
contaminant. In this context, it is important for
microbiologists as well as infectious disease
specialists to be aware that a poorly growing
aerobic gram-positive rod may be an aerotolerant
anaerobe and a pathogen like C. tertium,
particularly in immunocompromised patients. 7
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CMPT Clinical Bacteriology Program February 2010 G094
In view of the unusual susceptibility pattern of
this organism and its resistance to most empiric
antibiotic combinations, an accurate diagnosis
of infection with this organism is important
for the choice of an appropriate antimicrobial
treatment. 6
REFERENCES
1. Tappe D, Dirks J, Muller R, et al. Fatal Clostridium
tertium septicemia in a nonneutropenic
patient. J Infect. 2005;50:76-80.
2. Leegaard TM, Sandven P, Gaustad P. Clostridium
tertium: 3 case reports. Scand J Infect
Dis. 2005;37:230-232.
3. Johnson EA, Summanen P, Finegold S. Clostridium.
In: Murray ea, ed. Manual of Clinical
Microbiology. Vol 2. 9th ed. ed. Washington,
DC.: ASM; 2007:889,903.
4. Gosbell IB, Johnson CG, Newton PJ, Jelfs J.
Clostridium tertium bacteremia: 2 cases and
review. Pathology. 1996;28:70-73.
5. Valtonen M, Sivonen A, Elonen E. A cluster
of seven cases of clostridium tertium septicemia
in neutropenic patients. Eur J Clin Microbiol
Infect Dis. 1990;9:40-42.
6. Thaler M, Gill V, Pizzo PA. Emergence of
Clostridium tertium as a pathogen in neutropenic
patients. Am J Med. 1986;81:596-600
7. Valtonen M, Sivonen A, Elonen E. A cluster
of seven cases of Closridium terutium septicemia
in neutropenic patient. Eur J Clin Microbiol
Infect Dise. 1990;9:40-42.
Cowan and Steel’s Manual for the Identification
of Medical Bacteria Third Edition GI Barrow and
RKA Feltham. 1993.
9. Farnum NR. 1994. Ch. 11. Acceptance sampling.
p. 305-361. Modern Statistical Quality
Control and Improvement. Duxbury Press, Belmont,
California.
Reporting diphtheroids:
While commonly used, the
terms “diphtheroids” and
coryneform bacteria and corynebacterium,
have taken on
a variety of subtle differences
in meaning. Corynebacterium
species has the most
specific interpretation. Coryneform
(a bacterium that
looks like a Corynebacterium)
species may include Listeria
species and Kurthia. 8
Of the three terms
“diphtheroids” has no taxonomic
significance but by
jargon is taken to denote skin
dwelling Corynebacterium
species, occasionally associated
with skin disease e.g.
trichomycosis), but generally
non-pathogenic. As such,
the term is often taken to
imply non-relevant or contaminating
flora. Use of the
term in the reporting of skin
infections as the one described
here may be misleading.
Table-2: G094 smear reported results – Bacterial component -
Reported results A B C C1 Total % Grade
11-50, >25, >50/oif, 2+/3+/4+ gram positive bacilli, +/- suggestive of
anaerobic bacilli +/- spores
52 17 11 13 93 70 4
4+ gram positive bacilli, suggestive of Clostridium species 1 1 1 4
heavy, 3+/4+ gram positive bacilli, suggestive of Clostridium/Bacillus
species
6 6 4 4
3+/4+ gram positive bacilli, suggestive of Clostridium/Bacillus/
Lactobacillus species
1 1 1 4
4+ gram positive bacilli, suggestive of Bacillus species 1 1 1 3
4+ gram positive bacilli, probable diphtheroids 1 1 1 3
2+/3+ gram positive bacilli, 3+/4+ gram negative bacilli, +/- 2+ gram
variable bacilli
5 3 8 6 4
1+ gram positive bacilli, 3+ gram negative bacilli, 1+ gram positive cocci 1 1 1 3
2+ gram positive cocci, 3+ gram negative bacilli, snnp 1 1 1 1
4+ gram positive bacilli, 1+ gram positive coccobacilli 1 1 1 3
3+/4+ gram variable bacilli 6 5 11 8 4
2+/3+ gram negative bacilli 3 2 5 4 3
refer 1 1 2 1 -
Total 76 28 13 15 132 100
snnp: sample not normally processed