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Journal of Clinical Microbiology, March 1999, p. 835-837, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Justification for Use of a Single Trichrome Stain
as the Sole Means for Routine Detection of Intestinal Parasites in
Concentrated Stool Specimens
James A.
Kellogg* and
Carol J.
Elder
Clinical Microbiology Laboratory, York
Hospital, York, Pennsylvania
Received 23 July 1998/Returned for modification 21 October
1998/Accepted 18 November 1998
 |
ABSTRACT |
Of 12,321 stool samples analyzed over a 6-year interval, 870 (7.1%) were positive for a total of 1,019 parasites, of which 1,011 (99.2%) were found in trichrome-stained smears of unconcentrated specimens while only 479 (47.0%) were detected in iodine-stained smears of concentrated samples. Stool specimens were next analyzed by
trichrome staining of both unconcentrated and concentrated specimens
preserved in either mercury-polyvinyl alcohol (PVA) or cupric PVA. Of
2,198 specimens, 171 (7.8%) were positive for a total of 208 parasites, 192 (92.3%) and 204 (98.1%) of which were found in the
unconcentrated and concentrated specimens, respectively (P < 0.05). In our patient population, examination of
a single trichrome-stained smear of a concentrated stool specimen is a cost-effective alternative to routinely analyzing both concentrated and
unconcentrated specimens for parasites.
| |
TEXT |
In the past, a thorough examination
of stool specimens for detection of parasites involved four microscopic
analyses: direct saline and iodine wet mounts of fresh (unpreserved)
stool, an iodine wet mount of a concentrated sample, and a
trichrome-stained smear of an unconcentrated sample (1, 4,
8). Because motile trophozoites cannot be detected in direct wet
mounts of preserved specimens, many laboratories are now routinely
performing only two microscopic analyses on stool specimens submitted
for detection of parasites: trichrome staining of the unconcentrated sample and iodine staining of a wet mount of the concentrated specimen
(2, 4, 14). In a continuing effort to maintain test
sensitivity while reducing material and labor costs as well as exposure
to toxic substances, including mercury and formaldehyde, there is an
increasing interest in reevaluating the efficiency of older microscopic
approaches and concepts, looking for more-rapid, less expensive,
potentially less toxic, and equally sensitive alternatives. In 1996, Hale et al. (9) documented the accuracy of using for
parasite detection a single, permanent, modified-trichrome-stained slide prepared from a concentrated specimen preserved in cupric polyvinyl alcohol (PVA).
The present study was performed in two phases and had two objectives.
The sensitivity in our patient population of parasite detection by
trichrome staining of unconcentrated specimens preserved in mercury-PVA
was retrospectively compared with that of iodine wet mounts of
concentrated specimens preserved in formalin. In addition, the
sensitivities of trichrome staining of unconcentrated and concentrated
specimens that had been preserved in either mercury-PVA or cupric PVA
were prospectively compared.
Retrospective determination of sensitivity of trichrome
staining of unconcentrated specimens versus that of iodine
wet mounts of concentrated specimens.
Prior to 1997, all
stool specimens submitted for ova and parasite analysis were inoculated
into a two-vial preservative kit (Meridian Diagnostics, Cincinnati,
Ohio). One vial contained mercuric chloride-PVA and the other
contained 10% buffered formalin. Trichrome staining was
performed on the unconcentrated specimen in mercury-PVA, and an iodine
wet mount of the concentrated specimen in formalin was done (4,
11). At least 200 to 300 fields of the trichrome-stained smears
were microscopically examined under 10× or 20× and then 100×
objectives for a total of approximately 8 min for each specimen. The
entire coverslip of each formalin smear was examined under the 10×,
40×, and 100× objectives for approximately 7 min.
Prospective comparison of sensitivities of trichrome stainings of
unconcentrated and concentrated specimens.
During 1997, each stool
specimen submitted for ova and parasite detection was inoculated into a
single vial containing either mercuric chloride-PVA (for approximately
12% of the patients) or copper sulfate-PVA (for about 88% of the
patients), as recommended by Hale et al. (9). A portion of
each PVA-preserved specimen was used to prepare a smear for trichrome
staining of the unconcentrated sample. The staining procedure used was
a modification of the Wheatly trichrome stain procedure incorporating
Hemo-De (1). Preserved specimens were then concentrated by a
saline-Hemo-De procedure (1, 9, 12) in which formalin was
replaced by saline and ethyl acetate was replaced by Hemo-De. Smears of
the concentrates were made by adding 2 drops of the centrifuged pellet of a specimen (resuspended in 0.9% saline) to 1 drop of
cupric PVA or mercury-PVA (depending on the chemicals in which the
specimen was received) on a glass slide. Smears were air dried at
room temperature overnight or at 37°C for 4 h. The
trichrome staining procedure was identical to that used for
unconcentrated-specimen smears. Trichrome-stained smears were
microscopically analyzed for smear adequacy and for the presence and
internal structure of parasites, using 10× or 20× and then 100×
objectives and scanning 200 to 300 fields for approximately 8 min.
The Z test for differences in proportions for independent
samples was used for statistical analysis of results
(13). A P value of <0.05 was selected
as the minimum level determining significance.
Detection of parasites in trichrome-stained smears of
unconcentrated specimens versus iodine wet mounts of concentrated
specimens.
From 13 February 1990 through 31 August 1996, 12,321 preserved specimens from a total of 9,366 patients were analyzed for ova and parasites. A total of 1,019 parasites were detected in 870 (7.1%) of the specimens from 767 (8.2%) of the patients. Of the
1,019 parasites found, 540 (53.0%), 8 (0.8%), and 471 (46.2%) were found only in trichrome-stained smears of unconcentrated specimens, only in iodine wet mounts of concentrated specimens, and in
both the unconcentrated-specimen trichrome-stained smears and the
concentrated-specimen smears, respectively (Table
1). Trichrome staining of unconcentrated
specimens permitted the detection of 99.2% (1,011 of 1,019) of the
total parasites found, while iodine wet mounts of concentrated
specimens allowed the detection of only 47.0% (479 of 1,019) of the
total parasites. This difference was significant (P < 0.001).
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TABLE 1.
Detection of parasites by trichrome staining of
unconcentrated specimens and by iodine staining of wet mounts of
concentrated specimens
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Determination of recovery of parasites by trichrome staining of
unconcentrated and concentrated specimens.
From 1 January 1997 through 31 December 1997, 2,198 preserved specimens from 1,623 patients
were analyzed for parasites. A total of 208 parasites were detected in
171 (7.8%) of the specimens and in 133 (8.2%) of the patients. Of 182 parasites detected after using cupric PVA, 167 (91.8%) were found in
trichrome-stained smears of the unconcentrated specimens while 178 (97.8%) were found in trichrome-stained smears of the concentrated
specimens (P < 0.05) (Table
2). Of 26 parasites detected when
mercuric PVA was used, 25 (96.2%) and 26 (100%) were found in
trichrome-stained smears of the unconcentrated and concentrated
specimens, respectively (not significant) (Table
3).
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TABLE 2.
Detection of parasites in trichrome-stained smears of
unconcentrated and concentrated specimens preserved in Cu-PVA
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TABLE 3.
Detection of parasites in trichrome-stained smears of
unconcentrated and concentrated specimens preserved in Hg-PVA
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The size, shape, and staining characteristics of each parasite found,
with the exception of
Blastocystis hominis, were
indistinguishable
regardless of whether smears were stained following
preservation
in cupric PVA or in mercury-PVA. When smears were stained
after
preservation with cupric PVA, the features of
B. hominis were
not as distinctive as they were after treatment with
mercury-PVA.
However, with experience, laboratory personnel could
reliably
detect this parasite when either fixative was used. Material
and
labor costs for processing and reading trichrome-stained smears
of
both a concentrated and an unconcentrated sample from each
patient were
$10.20, or $22.420 for 2,198 specimens processed
during the course of
the year. Reading only the single trichrome-stained
smears of
concentrated specimens would have resulted in a savings,
during the
year, of $11,210.
The retrospective analysis of 6 1/2 years of recovery data indicated
that in our patient population, routine use of both an
iodine-stained
smear prepared from the formalin concentrate and
a trichrome-stained
direct smear (the two methods currently recommended
for routine
detection of parasites [
4,
11]) was not
cost-effective.
Garcia et al. (
3) have reported similar
findings. In their
study, 2,204 (99.8%) and only 644 (29.2%) of
pathogenic protozoa
were detected by trichrome staining of
unconcentrated samples
and wet mounts of formalin-ether concentrates,
respectively. Those
authors showed that both the cyst and
trophozoite forms of
Entamoeba histolytica,
Entamoeba
hartmanni, and
Giardia lamblia, as well
as the
trophozoite form of
Dientamoeba fragilis, were easily missed
if the laboratory examination was limited to only a microscopic
analysis of a wet mount of the concentrated sediment. On the basis
of
the results of the present study, this laboratory no longer
routinely
collects stool specimens in formalin but relies on single
specimens
that are individually preserved in a vial of
PVA.
During our prospective study of parasite detection by trichrome
staining of specimens preserved in either cupric or mercuric
PVA, our
rates of detection (7.8% of the specimens and 8.2% of
the patients
were positive) were almost identical to our rates
of detection in prior
years, when both trichrome staining of unconcentrated
samples and
iodine wet mounts of concentrated specimens were routinely
performed
(7.1% of the specimens and 8.2% of the patients were
positive during
the earlier interval). Furthermore, of the 208
parasites detected
during the prospective study, 204 (98.1%) were
found in
trichrome-stained smears of concentrated specimens while
only 192 (92.3%) were detected by similar staining of the unconcentrated
samples. Therefore, a single trichrome-stained smear of a
concentrated
specimen, as suggested by Hale et al. (
9),
appears to offer
an acceptably sensitive, cost-effective alternative to
processing
and microscopically analyzing both unconcentrated and
concentrated
stool specimens from our patient
population.
Advantages of processing only one slide, a trichrome-stained smear of a
concentrated stool specimen, when routine ova and
parasite detection is
ordered include the use of only one transport
vial per specimen
(instead of two); the elimination of toxic chemicals,
including mercury
(if cupric PVA is used) and formalin; increased
detection of parasites
and a greater number of organisms per positive
slide when the
concentrated sediment is stained instead of the
unconcentrated smear;
the availability of a permanently stained
slide that may be filed; and
the obvious cost savings, in both
material and labor, when only one
slide per specimen is stained
and analyzed (
9).
Some parasites, such as
Isospora belli,
Cryptosporidium spp., and
Cyclospora spp., may
stain poorly by the modified trichrome
stain procedure. Examination
of a modified-acid-fast-stained smear
of the PVA concentrate may be
necessary to confirm the presence
of these parasites. Hale et al.
(
9) reported that helminth
ova with thick outer shells
were easily identified by using the
modified trichrome stain. For
helminth ova with thin shells, including
Hymenolepis spp.
and
Schistosoma spp., examination of an iodine
wet mount of
the PVA concentrate aided in species identification.
One previous study
reported that 14.6% of helminth ova or larvae
were detected only by
trichrome staining of concentrated specimens
(
15). Some
parasites, including
G. lamblia, hookworm eggs, and
Trichuris eggs, may not concentrate as well from specimens
preserved
in PVA as from those preserved in formalin (
4).
Laboratory
personnel should be aware of the prevalence of parasites in
their
own populations and select their diagnostic methods
accordingly.
Copper sulfate has been added to Schaudinn's fixative as a less toxic,
more environmentally compatible alternative to mercuric
chloride. Its use for detection of parasites, however, has been
controversial. In 1981, Horen (
10) reported that smears
containing
protozoa fixed with copper sulfate (but not cobalt chloride)
exhibited
satisfactory nuclear and cytoplasmic morphological detail
following
trichrome staining. Hale et al., in 1996, found that protozoa
exhibited less shrinkage and more-distinct internal structure
with
trichrome staining after saline concentration of cupric-PVA-preserved
specimens, compared to the morphology observed in stained smears
of
unconcentrated specimens in cupric PVA (
9). However, Garcia
et al. (
6) reported in 1983 that the overall morphology of
protozoa preserved in PVA with copper sulfate was not as good
as that
achieved when the protozoa were preserved in PVA with
mercuric
chloride. In the present study, there was no apparent
difference in the
background colors or the morphological or staining
characteristics of
protozoa detected when copper was used instead
of mercury, with the
exception of one species:
B. hominis. The
differing
results reported when copper sulfate-containing fixatives
were used
could be a function of their sources. Commercially available
copper
sulfate-PVA today, at least that from the source used during
the
present study, appears to be an adequate, although not perfect,
alternative to mercuric chloride-PVA.
Other mercury-free fixatives that have been recently reported to
perform satisfactorily include zinc sulfate-PVA for trichrome-stained
smears (
7) and EcoFix for both trichrome- and
EcoStain-stained
smears (
5). Many of these alternatives may
result in some staining
of protozoa which lacks the color intensity or
precise nuclear
and cytoplasmic detail seen when the organisms
are stained following
fixation in mercuric chloride. As long as these
alternatives permit
accurate, reliable detection and
identification of the parasites,
however, laboratory personnel
may have to accept the fact that
protozoa may not always be as
crisply stained as they were when
mercuric chloride was used
(
7), and with experience, they will
most likely become
accustomed to small differences in staining
characteristics.
Routine laboratory tests designed to detect and identify parasitic
pathogens should be selected after taking into consideration
the
prevalence of different pathogens in the specific laboratory's
population. In some parts of the United States with large populations
of immigrants or AIDS patients, a single trichrome-stained smear
of a
concentrated specimen may not adequately detect parasites
that normally
cause disease in those patients. In our population
in central
Pennsylvania, as well as in that of Hale et al. (
9)
(Salt
Lake City), the routine use of an iodine wet mount prepared
from a
formalin-Hemo-De concentrate was not cost-effective. In
both
populations, the use of a single trichrome-stained smear
of a
concentrated stool specimen preserved in cupric PVA offered
very
acceptable sensitivity for routine parasite detection as
well as a
substantial savings in terms of materials and labor.
Performance of
iodine wet mounts or modified acid-fast staining
of the concentrated
specimens when necessary and appropriate permits
the detection or
confirmation of suspected parasites, including
helminths,
I. belli,
Cryptosporidium spp., and
Cyclospora spp.
| |
ACKNOWLEDGMENTS |
Statistical analysis of the results was performed by Sally
Cavanaugh, York Hospital Department of Research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Clinical
Microbiology Laboratory, York Hospital, 1001 S. George St.,
York, PA 17405. Phone: (717) 851-2393. Fax: (717) 851-2707. E-mail: jkellogg{at}yorkhospital.edu.
| |
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Journal of Clinical Microbiology, March 1999, p. 835-837, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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