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Previous Article | Next Article 
Journal of Clinical Microbiology, June 1998, p. 1814-1818, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Blinded, Externally Controlled Multicenter
Evaluation of Light Microscopy and PCR for Detection of
Microsporidia in Stool Specimens
Heinz
Rinder,1,*
Klaus
Janitschke,2
Horst
Aspöck,3
Alexandre J.
Da
Silva,4
Peter
Deplazes,5
Daniel P.
Fedorko,6
Caspar
Franzen,7
Ursula
Futh,8
Frank
Hünger,9
Anselm
Lehmacher,10
Christian G.
Meyer,11
Jean-Michel
Molina,12
Jörg
Sandfort,13
Rainer
Weber,14
Thomas
Löscher,1 and
the
Diagnostic Multicenter Study Group on Microsporidia
Department of Infectious Diseases and Tropical Medicine,
University of Munich, Munich,1
Robert
Koch Institute,2
Auguste Viktoria
Hospital,8
Institute for Tropical
Medicine, Humboldt University,11 and
Virchow Clinic, Medical Polyclinic,13
Berlin,
Clinic I for Internal Medicine, University of Cologne,
Cologne,7 and
Bernhard Nocht
Institute,9 and
Department of
Bacteriology, Institute of Hygiene,
Hamburg,10 Germany;
Institute of Hygiene, University of Vienna,
Vienna, Austria3;
Centers for Disease
Control and Prevention, Atlanta, Georgia 303414;
National Institutes of Health, Bethesda, Maryland
208926;
Institute of Parasitology,
University of Zurich,5 and
Division of
Infectious Diseases and Hospital Epidemiology, University
Hospital,14 Zurich, Switzerland; and
Department of Infectious Diseases, Hospital
Saint-Louis, University of Paris VII, Paris,
France12
Received 19 December 1997/Returned for modification 13 February
1998/Accepted 20 March 1998
 |
ABSTRACT |
The quality parameters for the detection of microsporidia in
identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where
technicians used PCR. The average overall sensitivities were 67% (89%
for patient samples only) for the PCR laboratories and 54% (80% for
patient samples only) for the light microscopy laboratories.
Specificities were 98 and 95%, respectively. Differences in results
were most apparent between the individual laboratories rather than
between the two major methods used.
| |
TEXT |
In-house evaluations of PCR
protocols, especially by those who developed them, are generally
satisfactory to excellent. In contrast, among the few blinded,
externally controlled evaluations of PCR protocols outside the field of
virology, there have been rather contrasting results. Impressive
examples are the studies by Noordhoek et al. of the detection of
Mycobacterium tuberculosis (17, 18). For
parasites, too, there often exist several PCR protocols for each of the
more frequently and many of the less frequently occurring
human-pathogenic species, including microsporidia (2-6, 8-10,
13-16, 20, 21, 23, 24). However, none of these protocols has
been validated in a blinded, externally controlled fashion. The only
exception is a study by five laboratories of solutions of
Toxoplasma gondii DNA (12). The various results from the different laboratories were attributed to the possible incompatibility of using DNA solutions instead of whole cells with some
of the DNA preparation methods. It was concluded that these artificial
samples were not appropriate for determining the PCR tests'
sensitivities and specificities for clinical specimens.
Hence, while one may recognize the potential of the PCR technique, the
probability of a positive result being correctly positive and a
negative result being from a parasite-free specimen is quite uncertain.
In an effort to assess the performance of this method for the detection
of a parasite, we have conducted a blinded, externally controlled,
multicenter study for the detection of microsporidia by PCR and
compared the results to those obtained by light microscopy. We have
chosen microsporidia (Enterocytozoon bieneusi,
Encephalitozoon intestinalis, Encephalitozoon
hellem, and Encephalitozoon cuniculi), intracellular,
spore-forming parasites responsible for diarrhea and other
manifestations in immunocompromised patients, because the small size of
the spores makes an alternative to their detection by light microscopy
especially advantageous.
Study design.
All technicians from 12 participating
laboratories were informed of and agreed to the following conditions.
Aliquots of 50 stool samples, approximately 1 g each, had to be
analyzed by each laboratory within 3 months. Only stool samples were
tested. Approximately 10 to 30 of the samples were to be negative
controls, most of them originating from healthy, immunocompetent
persons, and the other samples had to be stool samples with confirmed
parasites other than microsporidia. Some positive samples were to be
from healthy persons and spiked with cultured spores of E. intestinalis, E. hellem, or E. cuniculi at
different concentrations. At least one-third of all samples had to be
duplicates, and all samples were assigned a random number. Specimens
were tested by light microscopy at six laboratories (laboratories M1 to
M6), and specimens were tested by PCR at the other six laboratories
(laboratories P1 to P6). The results were to be made public
independently of the outcome, with the participating laboratories
remaining anonymous.
Stool samples containing spores identified as Enterocytozoon
spp. by light or electron microscopy or both were from seven immunocompromised patients diagnosed with microsporidiosis. The species
was confirmed to be Enterocytozoon bieneusi by sequencing of
the internal transcribed spacer of the rRNA gene (19). The samples were stored at 
20°C until they were divided into aliquots. Spores from Encephalitozoon species (E. cuniculi
IPZ:CH-H14, E. hellem IPZ:CH-H3, and E. intestinalis IPZ:D-H11) were isolated, cultivated on human
embryonic lung fibroblast (MRC-5) cells, characterized both
phenotypically and genotypically as described by Deplazes et al.
(7), and stored at 20°C until used for spiking. Negative stool samples, including those to be spiked, were from immunocompetent, healthy persons without travel histories for the prior 3 months. Four
stool samples were from immunocompetent outpatients suffering from
diarrhea who had confirmed infections with (i) Blastocystis hominis, Endolimax nana, and Entamoeba
hartmanni; (ii) Giardia lamblia only; (iii)
Heterophyes heterophyes only; or (iv) Entamoeba histolytica and B. hominis. Aliquots were prepared,
coded, and distributed by and the results were received at the Robert
Koch Institute in Berlin, Germany, which did not participate in the sample analyses. On the day of the sample preparation, the negative controls were prepared first, the patient samples were next, and the
spiked samples were last. For the spiked samples, the aliquots were
spiked individually. Utmost care was taken to avoid cross-contamination during sample preparation. All samples were shipped without fixatives and at ambient temperature by mail or overseas courier service.
Laboratories were asked to decide on the presence or absence of
microsporidia in every sample only by the method assigned to them
(either light microscopy or PCR) and to use their own protocols for
analysis. If possible, they should specify the genus and the species of
the microsporidia present. The methods used are summarized in Table
1 for laboratories M1 to M6 and in Table 2 for laboratories P1 to P6.
Sensitivity.
For the six PCR laboratories, the average
sensitivity with all samples was 67% (89% for the patient samples
only and 44% for the spiked samples only), with a range of 36 to 96%.
For the six light microscopy laboratories, the average sensitivity with
all samples was 54% (80% for the patient samples only and 27% for the spiked samples only), with a range of 25 to 71%. In this study, differences in sensitivity were more dependent on the individual laboratory than on the analysis method (Table
3). All things considered, technicians at
the PCR laboratories achieved, on average, a higher sensitivity (67%)
than those at the light microscopy laboratories (54%), scoring 9 percentage points better with the patient samples and 17 percentage
points better with the spiked samples. But these averages obscure the
wide variations in results between the individual laboratories and must
not be taken as the sole argument for favoring one method over the
other. For example, 100% sensitivity with the patient samples was
attained at five laboratories, three of which were light microscopy
laboratories. With samples spiked at the highest concentration,
106 spores/g, technicians from all but one microscopy
laboratory detected four or more of the six samples, results which
could be matched at only three of the PCR laboratories (Table
4). Light microscopy therefore appears to
be the more robust method for high concentrations of spores, while PCR
is more sensitive for low concentrations of spores.
The average sensitivity was higher at laboratories where nested PCRs
were performed (75%) than at those where single PCRs were performed
(63%), but again, variation between individual laboratories was high
and the best sensitivity was attained at a laboratory where only single
PCRs were performed (P1). However, the most extensive sample
preparation, including a spore concentration step starting with 1 g of stool, occurred at this laboratory. Except for this laboratory, a
possible dependency on the method of DNA isolation was not obvious. For
example, technicians at laboratory P2, which had the second-best
overall score and a higher specificity than that attained at P1, used a
very simple sample preparation requiring not more than 1 h to
complete (Table 2). Similarly, there was no correlation between the
number of repetitions of analyses per sample and the achieved
sensitivities. Surprisingly, the same was true for checking for PCR
inhibitors (Table 2), although this was just one of several differences
between the PCR protocols, but probably not the most decisive one.
Specificity.
Specificity was high at all laboratories, i.e.,
either 95 or 100%, except at one microscopy laboratory which scored
77% (Table 3). With the exception of this laboratory, no laboratory
reported more than one false-positive result, regardless of the method used. This came as a surprise at least for the PCR technique, where
cross-contaminations are an imminent danger. In contrast to results of
earlier surveys (17, 18), PCR contaminations were infrequent
in this study and occurred both in laboratories where single PCRs were
performed (P1 and P6) and in one where nested PCRs were performed (P3).
Still, more than one such result was never encountered in any
laboratory.
Detection limit.
A uniform detection limit between
104 and 106 spores per g of stool was apparent
for light microscopy only (Table 4). While technicians at two PCR
laboratories detected concentrations as low as 102
spores/g, most of the individual results of PCR were varied and increasing spore concentrations did not per se lead to higher sensitivities, except at one laboratory, P1, where all spiked samples
were correctly identified but where one sample was also falsely
determined to be positive. While samples containing 104 or
fewer spores per g could be detected, with a single exception, by PCR
only, four or more of the six samples spiked at high concentrations of
106 spores/g were detected by technicians at all but one
light microscopy laboratory. Only three PCR laboratories could match
this result. Therefore, detection limits were lower but inconsistent by
PCR, and light microscopy proved to be the more robust method for
detecting high concentrations of spores.
In view of these data, it can be speculated that a substantial number
of microsporidial infections currently go undetected, as has been
previously suspected for E. intestinalis infections (11), especially with, but not limited to, the
moderate-to-low concentrations of spores which might be found in less
severely immunocompromised patients. The high sensitivity obtained with the patient samples does not necessarily contradict this explanation, since these samples were screened by light microscopy and are therefore
biased towards high concentrations of spores.
Genus and species differentiation.
Genus determination, which
was attempted by technicians at two of the six light microscopy
laboratories and at five of the six PCR laboratories, was correctly
done in all cases. This finding is therapeutically relevant, because
only Encephalitozoon spp. are susceptible to benzimidazole
treatment. Morphologically, the spores can be differentiated by size;
Enterocytozoon bieneusi spores measure between 1 and 1.5 µm, and Encephalitozoon spp. measure between 2 and 3 µm.
The species cannot be distinguished by spore morphology. Technicians at
five of the six PCR laboratories differentiated the microsporidia to
the species level by analyzing the PCR products and were successful in
87 to 100% of all attempts. Accuracy was highest (89 to 100%) when
species-specific primers were used (Table
5). The use of restriction fragment
length polymorphism (RFLP) at laboratory P2 was slightly less accurate
(87%). However, the percentage of samples correctly identified to the
species level by RFLP (71%) was almost identical to the average
percentage (73%) from laboratories P1, P3, and P4, where
species-specific primers for all four species were used (Table 5).
Practical conclusions.
The apparent differences in the quality
of results between laboratories rather than between the methods
employed might be considered an argument for establishing centralized
reference centers to guarantee a high level of quality in the detection of microsporidia in stool samples. Until that time, individual laboratories are encouraged to improve their respective techniques as
much as possible but not to give up one method in favor of the other.
It will certainly be helpful to have a second technique available when
a confirmation of the result of the first is desired. While microscopy
is known to be highly dependent on the expertise of the examiner, it is
not certain if, on the other hand, the generation of a standardized PCR
test kit will be a more promising way to guarantee reliable detections
of microsporidia by a larger number of laboratories. From the only
prior bacteriological study which addressed this question, an
unexpected result was that the use of commercial PCR kits did not
produce better results than in-house PCR protocols (18). If
the reliable detection of microsporidia is not to be restricted to a
limited number of reference centers, techniques other than light
microscopy and PCR, e.g., coproantigen enzyme-linked immunosorbent
assays, might have to be developed.
| |
ACKNOWLEDGMENTS |
This study would not have been possible without the collaboration
and expertise of Cüneyt Acar, Ingrid Blöschl, Christiane Bug, Nico P. Kock, Sabine Köhler, Manuela Reisig, Bärbel
Sauer, Justus Schottelius, Slavomir Sproski, Isabelle Tanner, and Ilse Veits.
This study was supported in part by a grant to J. M. Molina from
SIDACTION, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Infectious Diseases and Tropical Medicine, Leopoldstrasse 5, D-80802 Munich, Germany. Phone: 49-89-21803618. Fax: 49-89-336112. E-mail: rinder{at}lrz.uni-muenchen.de.
Herbert Auer, Felicia David, Bernhard Fleischer, Andreas Haßl,
Walter Heise, Thomas Hoppe, Olivier Liguory, Andreas Müller, Nancy A. Nelson, Otto Picher, Norman J. Pieniazek, and Angelika Thomschke.
| |
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Journal of Clinical Microbiology, June 1998, p. 1814-1818, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Franzen, C., Muller, A.
(1999). Molecular Techniques for Detection, Species Differentiation, and Phylogenetic Analysis of Microsporidia. Clin. Microbiol. Rev.
12: 243-285
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