JCM Accepts, published online ahead of print on 26 November 2014 J. Clin. Microbiol. doi:10.1128/JCM.02836-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Multi-site clinical evaluation of a rapid test for Entamoeba histolytica in stool
2 3 4
Hans P. Verkerkea , Blake Hanburyb, Abdullah Siddiquec, Amidou Samied, Rashidul
5
Haquec, Joel Herbeinb, and William A. Petri Jr. a#
6 7
Division of Infectious Diseases and International Health, University of Virginia,
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Charlottesville, Virginia, USAa; TECHLAB, Blacksburg, Virginia, USAb; Parasitology,
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International Center for Diarrheal Disease Research, Dhaka, Bangladeshc;; University
10
of Venda, Limpopo province, South Africad
11 12
Running Head: Clinical evaluation of the E. HISTOLYTICA QUIK CHEK
13 14 15 16 17 18 19 20 21 22
#Address correspondence to William A. Petri Jr., [email protected]
23
Abstract
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Rapid, point-of-care detection of enteric protozoa in diarrheal stool is
25
desirable in clinical and research settings to efficiently determine the etiology of
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diarrhea. We analyzed the ability of the third-generation E. HISTOLYTICA QUIK
27
CHEK™ developed by TECHLAB to detect amebic antigen in fecal samples from
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independent study populations in South Africa and Bangladesh. We compared the
29
performance of this recently released rapid test to those of the commercially
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available ProSpecTTM Entamoeba histolytica microplate assay from Remel and the E.
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HISTOLYTICA II ELISA from TECHLAB, using real-time and nested PCR for
32
Entamoeba species to resolve discrepant results. After discrepant resolution, The E.
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HISTOLYTICA QUIK CHEK™ exhibited sensitivity and specificity compared to
34
the E. HISTOLYTICA II ELISA of 98.0 percent (95% CI: 92.9%-99.8%) and 100
35
percent (95% CI: 99.0%-100%) respectively. Compared to the ProSpecT™
36
microplate assay the QUIK CHEK exhibited 97.0 percent (95% CI: 91.5%-99.4%)
37
sensitivity and 100 percent (95% CI: 99.0%-100%) specificity. Our results indicate
38
that the QUIK CHEK is a robust assay for specific detection of Entamoeba histolytica
39
trophozoites in un-fixed frozen clinical stool samples.
40 41
Introduction
42
Entamoeba histolytica is a protozoan parasite that invades through the
43
intestinal epithelium via a unique cell biologic process called trogocytosis, resulting
44
in diarrhea, dysentery and extraintestinal disease (1, 2). E. histolytica exhibits an
45
oral-fecal transmission pattern and is endemic to resource-limited communities of
46
South and Central America, Asia, Africa, Mexico, and the Pacific Islands (reviewed in
47
3, 4). Our longitudinal studies of urban populations in Bangladesh have linked E.
48
histolytica infection with childhood morbidities, including stunting and delayed
49
cognitive development (5–7). Furthermore, the prospective case-control “Global
50
Enteric Multicenter Study” (GEMS) recently identified E. histolytica among the top
2
51
ten agents of moderate to severe diarrhea in two of their seven study sites across
52
Africa and South East Asia. Across all seven study sites, E. histolytica was associated
53
with the greatest risk of mortality in the second year of life and more common in
54
infants with severe diarrhea (8). In industrialized countries, travelers from endemic
55
regions and men who have sex with men are at higher risk than the general
56
population of acquiring E. histolytica infections (9–11). These and many other
57
observations support the importance of detecting E. histolytica in stool and
58
controlling transmission in affected populations.
59
Aside from E. histolytica, a complex of morphologically indistinguishable
60
Entamoeba species colonizes the human intestine. Entamoeba dispar and the free-
61
living Entamoeba moshkovskii have traditionally been classified as non-pathogenic.
62
And though specific strains of E. dispar have been associated with human colitis and
63
amebic liver abscess, these results have not been independently replicated (12–14).
64
E. moshkovskii though may be a cause of non-invasive diarrhea (15). And the
65
recently discovered Entamoeba bangladeshi is of unknown virulence (16). Thus E.
66
histolytica is the only Entamoeba species known to be capable of causing invasive
67
intestinal disease. However infection with other Entamoeba species complicates
68
diagnosis. And their presence calls for continued development and validation of
69
effective diagnostic tools with reliable Entamoeba species resolution.
70
Conventional detection of E. histolytica infection is by fecal microscopy,
71
which suffers from low sensitivity and specificity. In recent years, single and
72
multiplex real-time PCR assays have replaced microscopy as gold standards of
73
detection for this parasite. At the same time, a number of microplate enzyme-linked
74
immunosorbent assays (ELISAs) and rapid immunochromatographic assays have
75
been developed to detect amebic antigen in stool. Available antigen detection
76
methods vary in their sensitivity and specificity and many cannot reliably
77
distinguish between E. histolytica and E. dispar.
78
The E. HISTOLYTICA II ELISA (TECHLAB, Blacksburg VA) uses monoclonal
79
antibodies against the E. histolytica Gal/GalNAc-specific lectin. It is the only FDA
3
80
approved commercially available microplate ELISA known to specifically detect E.
81
histolytica and exclude infection by other Entamoeba species (19). This ELISA
82
exhibits greater sensitivity than the combination of microscopy and culture but has
83
been reported to exhibit lower sensitivity (79%) and specificity (96%) relative to
84
real-time PCR (20). The remel ProSpecT™ E. histolytica microplate assay (THERMO
85
FISCHER, USA) also exhibits greater sensitivity than microscopy and culture, but it is
86
known to cross-react to some extent with E. dispar.
87
Here we report a multi-site comparative analysis of the third generation E.
88
HISTOLYTICA QUIK CHEK™ with the E. HISTOLYTICA II ELISA and ProSpecT™ E.
89
histolytica microplate assay. We demonstrate in stool samples from Bangladesh and
90
South Africa that this rapid immunochromatographic cassette exhibits robust
91
detection of E. histolytica relative to the existing microplate assays. The decreased
92
turn around time, simple platform, and easy interpretation of rapid tests such as the
93
QUIK CHEK offer significant advantages in clinical settings, allowing a health care
94
provider to provide point-of-care diagnosis and treatment.
95
Methods
96
Study populations and clinical samples
97
Frozen clinical stool specimens from South Africa were collected for a study
98
of opportunistic infections among patients at risk of HIV infection in Limpopo
99
province. Testing was authorized by the Health, Research and Ethical committee of
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the University of Venda. Clinical diarrheal and surveillance stool specimens from
101
Dhaka, Bangladesh were obtained in a prospective study of infants and children
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living in a slum community. 310 samples were obtained from the ICDDR,B sample
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repository and 148 from the University of Venda. Testing was first authorized by the
104
Research Review and Ethical Review Committees of the International Center for
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Diarrheal Disease Research, Bangladesh (ICDDR,B) and the Institutional Review
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Board of the University of Virginia. 1 sample from the ICDDR,B and 8 samples from
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South Africa had insufficient material for testing. 160 of the samples from the
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ICDDR,B population were tested at TECHLAB. All remaining samples with sufficient 4
109
material for all three tests were tested at the ICDDR,B. Frozen un-fixed stool
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samples were shipped on dry ice to maintain a cold chain in transit. As part of the
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sample intake procedure, fresh unfrozen stool samples were assessed by
112
microscopy for protozoan parasites. These archival data were used in part to enrich
113
our sample pool in samples likely to contain Entamoeba species.
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Each specimen was tested by E. HISTOLYTICA QUIK CHEK™ (TECHLAB,
115
Blacksburg, Virginia), E. HISTOLYTICA II ELISA (TECHLAB, Blacksburg, Virginia), and
116
Remel ProSpecT ™ ELISA (THERMO FISCHER, USA). Discrepant results were tested
117
by real-time PCR for E. histolytica and E. moshkovskii and a nested PCR for E. dispar.
118
Detection of E. histolytica by E. HISTOLYTICA QUIK CHEK™
119
All reagents and specimens were brought to room temperature prior to
120
testing. 25 l of liquid stool or a small 2 mm diameter portion of solid stool was
121
homogenized with 500 l of diluent premixed with 1 drop of conjugate. External
122
controls were prepared for each set of samples by adding one drop of positive
123
control or 25 l diluent to 500 l diluent. 500 l of sample or control was added to
124
the sample well of a membrane device and incubated for 15 minutes. 300 l of wash
125
buffer followed by 2 drops of substrate were added directly to the reaction window.
126
Internal control (C) and test (T) lines were read after 10 minutes. Figure 1 depicts
127
examples of negative and positive results on the QUIK CHEK.
128
Detection of E. histolytica by E. HISTOLYTICA II and ProSpecT™ microwell
129
ELISAs
130
Frozen liquid and solid stool specimens were handled and shipped according
131
to the kit manufacturers’ instructions. Testing was performed according to the
132
instructions of the manufacturer for both ELISA kits. Interpretation for both assays
133
was based on a single OD reading at 450 nm with negative control subtracted OD
134
values greater than or equal to 0.050 considered positive for E. histolytica.
5
135
Resolution of discrepant results by Entamoeba species-specific polymerase
136
chain reaction
137
Samples that were discrepant by at least one of the three antigen detection
138
methods were tested by PCR for E. histolytica, E. dispar, and E. moshkovskii. The E.
139
histolytica real-time PCR assay was performed as part of a multiplex assay including
140
Giardia intestinalis and Cryptosporidium spp.. according to the protocol described by
141
Haque et al. (17). A 2-step nested PCR was performed to detect E. dispar according
142
to the protocol described by Haque et al. (18). An in-house TaqMan assay was used
143
to detect E. moshkovskii (Em-f GCG CAA GCT AAG TTT CTA GGA ATG AGG; Em-r TCC
144
TTT TAA TCC TTC TCT CGA AAT GTC CGAA; Em-P HEX-CGA AGG AGA TGA AGT GAG
145
TAA TCA CTT TATC –BHQ1). Reaction conditions were briefly as follows: 2 mmol/L
146
MgCl2, 0.4 mol/L each of forward and reverse primers, 0.08 mol/L of probe, 3 L
147
of extracted DNA, and Qiagen master mix with a final reaction volume of 25 l. The
148
cycling conditions consisted of 15 minutes at 95°C followed by 40 cycles of 15
149
seconds at 95°C and 1 minute at 58°C.
150
Results
151
458 samples were collected for the study. 449 samples had sufficient
152
material and were tested by the E. HISTOLYTICA QUIK CHEK™, E. HISTOLYTICA II
153
ELISA, and ProSpecT™ Entamoeba histolytica microplate assay. 9 samples were
154
excluded from the analyses because of insufficient material. Age and gender data
155
were available for 350 and 424 samples respectively. 87% of the samples came from
156
participants less than 18 years of age. On average, the samples were derived from
157
populations that were 46% male and 54% female.
158
Comparative analysis of the E. HISTOLYTICA QUIK CHEK™ assay for rapid
159
detection of E. histolytica in stool.
160
84 samples were positive and 327 were negative by all three detection
161
methods. 3 samples positive by QUIK CHEK and 3 samples negative by QUIK CHEK
162
were discrepant with the E. HISTOLYTICA II ELISA. While 10 samples positive and
6
163
24 samples negative by QUIK CHEK were discrepant with the ProSpecT™ E.
164
histolytica ELISA. These 40 instances of discrepancy were attributable to 38 child
165
stool samples. 2 samples were discrepant in both analyses. The 38 samples
166
discrepant in either of the two comparative analyses were subsequently resolved by
167
PCR for E. histolytica, E. dispar, and E. moshkovskii.
168
Table 1 consists of two 2 x 2 tables comparing the E. HISTOLYTICA QUIK
169
CHEK™ to the E. HISTOLYTICA II ELISA (a) and the ProSpecT™ E. histolytica ELISA
170
(b). The E. HISTOLYTICA
171
99.4%) sensitivity and 99.2 percent (95% CI: 97.5%-99.8%) specificity compared
172
with the E. HISTOLYTICA II ELISA. The QUIK CHEK exhibited 78.4 percent (95% CI:
173
69.6%-85.6%) sensitivity and 97.0 percent (95% CI 94.6%-98.6%) specificity
174
compared with the ProSpecT™ E. histolytica ELISA. Overall, the E. HISTOLYTICA QUIK
175
CHEK™ exhibited the strongest correlation with the E. HISTOLYTICA II ELISA results
176
prior to resolution by species specific PCR.
177
Resolution of discrepant results by Entamoeba species specific polymerase
178
chain reaction
QUIK CHEK demonstrated 96.9 percent (95% CI: 91.2%-
179
Table 2 consists of two 2 x 2 tables comparing the E. HISTOLYTICA QUIK
180
CHEK™ to the E. HISTOLYTICA II ELISA with discrepant results resolved by PCR for
181
E. histolytica (a) and to the ProSpecT™ E. histolytica ELISA with discrepant results
182
resolved by PCR for E. histolytica (b). 38 samples were discrepant by one of the
183
three antigen detection tests. After PCR resolution of discrepant results between the
184
two assays, the E. HISTOLYTICA QUIK CHEK™ exhibited sensitivity and specificity
185
compared to the E. HISTOLYTICA II ELISA of 98.0 percent (95% CI: 92.9%-99.8%)
186
and 100 percent (95% CI: 99.0%-100%) respectively. The same discrepant
187
resolution yielded sensitivity and specificity values of 97.0 percent (95% CI: 91.5%-
188
99.4%) and 100 percent (95% CI: 99.0%-100%) for the E. HISTOLYTICA QUIK
189
CHEK™ compared to the ProspecT™ E. histolytica ELISA.
190
In order to identify possible sources of amebic cross reactivity, samples with
191
discrepant results were also analyzed by nested PCR for E. dispar and real-time PCR 7
192
for E. moshkovskii. No samples tested positive by PCR for E. moshkovskii. 13 samples
193
tested positive for E. dispar. These E. dispar positive samples all tested positive for E.
194
histolytica by ProSpecT™ ELISA and negative for E. histolytica by the E. HISTOLYTICA
195
QUIK CHEK™, E. HISTOLYTICA II ELISA and E. histolytica-specific PCR. All other
196
discrepant samples tested negative for E. dispar. These data suggest that the
197
ProSpecT™ ELISA may cross-react to some degree with E. dispar.
198
In a supplementary analysis, we tested 9 additional clinical stool samples
199
from Bangladesh, which were screened by PCR for single Entamoeba species. 3
200
samples were positive for E. histolytica and negative for E. dispar and E. moshkovskii.
201
3 samples were positive for E. dispar and negative for E. histolytica and E.
202
moshkovskii. And 3 samples were positive for E. moshkovskii and negative for E.
203
histolytica and E. dispar. The 3 E. histolytica positive samples tested positive by E.
204
HISTOLYTICA QUIK CHEK™. E. dispar and E. moshkovskii positive samples did not
205
cross-react with the E. HISTOLYTICA QUIK CHEK™ (Data not shown). These
206
preliminary data suggest that the E. HISTOLYTICA QUIK CHEK™ is unlikely to cross-
207
react with E. dispar or E. moshkovskii.
208
Discussion
209
The major finding of our multi-site clinical evaluation of the E. HISTOLYTICA
210
QUIK CHEK™ is that this rapid detection method demonstrates comparable
211
performance with two ELISA-based E. histolytica antigen detection methods.
212
Furthermore, we demonstrate through PCR analysis of discrepant results, a high
213
rate of E. histolytica false positivity for the ProSpecT ™ E. histolytica ELISA relative to
214
the other methods used. This observation may in part be explained by cross
215
reactivity of this test with nonpathogenic E. dispar, which is supported by the
216
presence of E. dispar DNA in more than half of the samples called positive for E.
217
histolytica by the ProSpecT™ ELISA and negative by other antigen detection
218
methods and PCR.
219
A previous study by Korpe et al., reported that the E. HISTOLYTICA QUIK
220
CHEK™ exhibited 100% sensitivity and specificity relative to the E. HISTOLYTICA II 8
221
ELISA (21). While our results are consistent with these findings, we expanded our
222
analysis to include comparison of the QUIK CHEK with the ProSpecT™ E. histolytica
223
ELISA and clinical stool specimens from populations in both Bangladesh and South
224
Africa.
225
Our results support the finding that the rapid E. HISTOLYTICA QUIK CHEK™
226
compares well for detection of E. histolytica in stool with the E. HISTOLYTICA II
227
ELISA. The majority of discrepant results in our study were attributable to the
228
ProSpecT™ E. histolytica ELISA. Using E. histolytica specific real-time PCR, we
229
demonstrated that 21 out of 38 discrepant samples (55.3%) were the result of
230
ProSpecT™ false positivity; 10 out of 38 (26.3%) resulted from ProSpecT™ false
231
negativity; while only 3 out of 38 (7.9%) resulted from QUIK CHEK false negativity; 4
232
out of 38 (10.5%) resulted from E. HISTOLYTICA II ELISA false negativity; and 1 out
233
of 38 (2.6%) resulted from E. HISTOLYTICA II ELISA false positivity. This analysis
234
was limited to the discrepant samples tested by PCR. We detected E. dispar in 13 out
235
of the 21 samples (61.9%) that were false positive by ProSpecT™ ELISA. When
236
compared to the E. HISTOLYTICA II ELISA, which is recommended by the Centers for
237
Disease Control (CDC) for specific diagnosis of E. histolytica infection, our results
238
demonstrate that the QUIK CHEK is a robust point-of-care assay to specifically
239
identify E. histolytica in stool specimens.
240
Our use of real-time PCR in this study was limited to discrepant resolution.
241
Future studies may seek to rigorously compare this rapid test with real-time PCR to
242
assess its true limits of detection. We also limited our species coverage in this study
243
to E. histolytica, E. dispar, and E. moshkovskii. While cross-reactivity with E. dispar is
244
likely to be the greatest concern in clinics and epidemiological studies, it will be
245
interesting in future studies to assess the reactivity of this assay with a wider range
246
Entamoeba species and perhaps other fecal microbes.
247 248
Acknowledgments
9
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This work was supported by NIH 5R01 AI043596-16 and a Fulbright
250
research grant to HPV. The authors acknowledge that Joel Herbein and Blake
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Hanbury are employed by TECHLAB and were involved in the development of both
252
the E. HISTOLYTICA QUIK CHEK™ assay and the E. histolytica II ELISA evaluated in
253
this manuscript. William A. Petri Jr. receives licensing fees from TECHLAB. These
254
fees are donated in full to the American Society for Tropical Medicine and Hygiene
255
(ASTMH).
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homosexual men: further evidence for the occurrence of pathogenic strains. Trans. R. Soc. Trop. Med. Hyg. 84:250–251. 10. Gunther J, Shafir S, Bristow B, Sorvillo F. 2011. Amebiasis-Related Mortality among United States Residents, 1990–2007. The American Journal of Tropical Medicine and Hygiene 85:1038 –1040. 11. Lim P-L, Han P, Chen LH, MacDonald S, Pandey P, Hale D, Schlagenhauf P, Loutan L, Wilder-Smith A, Davis XM, Freedman DO, GeoSentinel Surveillance Network. 2012. Expatriates ill after travel: results from the Geosentinel Surveillance Network. BMC Infect. Dis. 12:386. 12. Dolabella SS, Serrano-Luna J, Navarro-García F, Cerritos R, Ximénez C, Galván-Moroyoqui JM, Silva EF, Tsutsumi V, Shibayama M. 2012. Amoebic liver abscess production by Entamoeba dispar. Ann Hepatol 11:107– 117. 13. Ximénez C, Cerritos R, Rojas L, Dolabella S, Morán P, Shibayama M, González E, Valadez A, Hernández E, Valenzuela O, Limón A, Partida O, Silva EF. 2010. Human Amebiasis: Breaking the Paradigm? Int J Environ Res Public Health 7:1105–1120. 14. Guzmán-Silva MA, Santos HLC, Peralta RS, Peralta JM, De Macedo HW. 2013. Experimental amoebic liver abscess in hamsters caused by trophozoites of a Brazilian strain of Entamoeba dispar. Exp. Parasitol. 134:39– 47. 15. Shimokawa C, Kabir M, Taniuchi M, Mondal D, Kobayashi S, Ali IKM, Sobuz SU, Senba M, Houpt E, Haque R, Petri WA Jr, Hamano S. 2012. Entamoeba moshkovskii is associated with diarrhea in infants and causes diarrhea and colitis in mice. J. Infect. Dis. 206:744–751. 16. Royer TL, Gilchrist C, Kabir M, Arju T, Ralston KS, Haque R, Clark CG, Petri WA Jr. 2012. Entamoeba bangladeshi nov. sp., Bangladesh. Emerging Infect. Dis. 18:1543–1545. 17. Haque R, Roy S, Siddique A, Mondal U, Rahman SMM, Mondal D, Houpt E, Petri WA Jr. 2007. Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. Am. J. Trop. Med. Hyg. 76:713–717. 18. Haque R, Ali IKM, Akther S, Petri WA. 1998. Comparison of PCR, Isoenzyme Analysis, and Antigen Detection for Diagnosis of Entamoeba histolytica Infection. J Clin Microbiol 36:449–452. 19. Haque R, Mollah NU, Ali IKM, Alam K, Eubanks A, Lyerly D, Petri WA. 2000. Diagnosis of Amebic Liver Abscess and Intestinal Infection with the TechLab Entamoeba histolytica II Antigen Detection and Antibody Tests. J Clin Microbiol 38:3235–3239. 20. Roy S, Kabir M, Mondal D, Ali IKM, Petri WA, Haque R. 2005. Realtime-PCR assay for diagnosis of Entamoeba histolytica infection. J. Clin. Microbiol. 43:2168–2172. 21. Korpe PS, Stott BR, Nazib F, Kabir M, Haque R, Herbein JF, Petri WA. 2012. Evaluation of a Rapid Point-of-Care Fecal Antigen Detection Test for Entamoeba histolytica. Am J Trop Med Hyg 86:980–981.
11
334 335 336 337 338 339 340 341 342 343
Tables and Figure legends
344 345
Figure 1. E. HISTOLYTICA QUIK CHEK™ negative and positive results. Left: a
346
negative result on the QUIK CHEK. Right: a positive result. C = control line (positive
347
internal control), T = test line (sample readout).
348 349 350 351 352 353 354 355 12
356 357 358 359 360 361 362 363 364 E. HISTOLYTICA II ELISA
a.
E. HISTOLYTICA QUIK CHEK™
Positive
Negative
Total
Positive
94
3
97
Negative
3
349
352
Total
97
352
449
QUIK CHEK™
Sensitivity 96.9 Specificity 99.2
95% CI 91.2 - 99.4
97.5 - 99.8
ProSpecT™E. histolytica ELISA
b.
E. HISTOLYTICA
%
Positive
Negative
Total
Positive
87
10
97
Negative
24
328
352
Total
111
338
449
% Sensitivity 78.4 Specificity 97.0
95% CI 69.7 - 85.6
94.6 - 98.6
13
365 366
Table 1. Comparative analysis of the E. HISTOLYTICA QUIK CHEK™ assay for
367
rapid detection of Entamoeba histolytica in stool. 449 frozen clinical diarrheal
368
and surveillance stool specimens were assayed to compare the performance of the
369
recently released E. HISTOLYTICA QUIK CHEK™ with those of two commercially
370
available microplate based methods for detection of E. histolytica in stool. Positive
371
and negative predictive agreements with each assay are reported with 95%
372
confidence intervals. (a) The QUIK CHEK exhibited 96.9% and 99.2% positive and
373
negative predictive agreement with the E. HISTOLYTICA II ELISA (TECHLAB®)
374
respectively. (b) The QUIK CHEK exhibited 78.4% and 97.0% positive and negative
375
predictive agreement with the ProSpecT™ E. histolytica ELISA (Remel).
376 E. HISTOLYTICA II ELISA or PCR
a.
E. HISTOLYTICA QUIK CHEK™
Positive
Negative
Total
Positive
97
0
97
Negative
2
350
352
Total
99
350
449
QUIK CHEK™
Sensitivity 98.0
95% CI 92.9 - 99.75
Specificity 100
99.0 - 100
ProSpecT™E. histolytica ELISA or PCR
b.
E. HISTOLYTICA
%
Positive
Negative
Total
Positive
97
0
97
Negative
3
349
352
Total
100
349
449
% Sensitivity 97.0 Specificity 100
95% CI 91.5 - 99.4
99.0 - 100
14
377 378
Table 2. Comparison of the E. HISTOLYTICA QUIK CHEK™ with two microwell
379
assays after real-time PCR resolution of discrepant results.
380
38 samples out of 449 tested resulted in 40 discrepancies when the E. HISTOLYTICA
381
QUIK CHEK™ was compared with two microwell ELISAs. All 38 samples were
382
assayed by the gold standard of detection, real-time PCR for E. histolytica. Sensitivity
383
and specificity compared with each assay after PCR resolution are reported with
384
95% confidence intervals. (a) The E. HISTOLYTICA QUIK CHEK ™ exhibited 98.0%
385
and 100% positive and negative predictive agreement with the E. HISTOLYTICA II
386
ELISA respectively. (b) The E. HISTOLYTICA QUIK CHEK™ exhibited 97.0% and
387
100% sensitivity and specificity compared with the Remel ProSpecT™ E. histolytica
388
ELISA.
15
1
Multi-site clinical evaluation of a rapid test for Entamoeba histolytica in stool
2 3 4
Hans P. Verkerkea , Blake Hanburyb, Abdullah Siddiquec, Amidou Samied, Rashidul
5
Haquec, Joel Herbeinb, and William A. Petri Jr. a#
6 7
Division of Infectious Diseases and International Health, University of Virginia,
8
Charlottesville, Virginia, USAa; TECHLAB, Blacksburg, Virginia, USAb; Parasitology,
9
International Center for Diarrheal Disease Research, Dhaka, Bangladeshc;; University
10
of Venda, Limpopo province, South Africad
11 12
Running Head: Clinical evaluation of the E. HISTOLYTICA QUIK CHEK
13 14 15 16 17 18 19 20 21 22
#Address correspondence to William A. Petri Jr., [email protected]
23
Abstract
24
Rapid, point-of-care detection of enteric protozoa in diarrheal stool is
25
desirable in clinical and research settings to efficiently determine the etiology of
26
diarrhea. We analyzed the ability of the third-generation E. HISTOLYTICA QUIK
27
CHEK™ developed by TECHLAB to detect amebic antigen in fecal samples from
28
independent study populations in South Africa and Bangladesh. We compared the
29
performance of this recently released rapid test to those of the commercially
30
available ProSpecTTM Entamoeba histolytica microplate assay from Remel and the E.
31
HISTOLYTICA II ELISA from TECHLAB, using real-time and nested PCR for
32
Entamoeba species to resolve discrepant results. After discrepant resolution, The E.
33
HISTOLYTICA QUIK CHEK™ exhibited sensitivity and specificity compared to
34
the E. HISTOLYTICA II ELISA of 98.0 percent (95% CI: 92.9%-99.8%) and 100
35
percent (95% CI: 99.0%-100%) respectively. Compared to the ProSpecT™
36
microplate assay the QUIK CHEK exhibited 97.0 percent (95% CI: 91.5%-99.4%)
37
sensitivity and 100 percent (95% CI: 99.0%-100%) specificity. Our results indicate
38
that the QUIK CHEK is a robust assay for specific detection of Entamoeba histolytica
39
trophozoites in un-fixed frozen clinical stool samples.
40 41
Introduction
42
Entamoeba histolytica is a protozoan parasite that invades through the
43
intestinal epithelium via a unique cell biologic process called trogocytosis, resulting
44
in diarrhea, dysentery and extraintestinal disease (1, 2). E. histolytica exhibits an
45
oral-fecal transmission pattern and is endemic to resource-limited communities of
46
South and Central America, Asia, Africa, Mexico, and the Pacific Islands (reviewed in
47
3, 4). Our longitudinal studies of urban populations in Bangladesh have linked E.
48
histolytica infection with childhood morbidities, including stunting and delayed
49
cognitive development (5–7). Furthermore, the prospective case-control “Global
50
Enteric Multicenter Study” (GEMS) recently identified E. histolytica among the top
2
51
ten agents of moderate to severe diarrhea in two of their seven study sites across
52
Africa and South East Asia. Across all seven study sites, E. histolytica was associated
53
with the greatest risk of mortality in the second year of life and more common in
54
infants with severe diarrhea (8). In industrialized countries, travelers from endemic
55
regions and men who have sex with men are at higher risk than the general
56
population of acquiring E. histolytica infections (9–11). These and many other
57
observations support the importance of detecting E. histolytica in stool and
58
controlling transmission in affected populations.
59
Aside from E. histolytica, a complex of morphologically indistinguishable
60
Entamoeba species colonizes the human intestine. Entamoeba dispar and the free-
61
living Entamoeba moshkovskii have traditionally been classified as non-pathogenic.
62
And though specific strains of E. dispar have been associated with human colitis and
63
amebic liver abscess, these results have not been independently replicated (12–14).
64
E. moshkovskii though may be a cause of non-invasive diarrhea (15). And the
65
recently discovered Entamoeba bangladeshi is of unknown virulence (16). Thus E.
66
histolytica is the only Entamoeba species known to be capable of causing invasive
67
intestinal disease. However infection with other Entamoeba species complicates
68
diagnosis. And their presence calls for continued development and validation of
69
effective diagnostic tools with reliable Entamoeba species resolution.
70
Conventional detection of E. histolytica infection is by fecal microscopy,
71
which suffers from low sensitivity and specificity. In recent years, single and
72
multiplex real-time PCR assays have replaced microscopy as gold standards of
73
detection for this parasite. At the same time, a number of microplate enzyme-linked
74
immunosorbent assays (ELISAs) and rapid immunochromatographic assays have
75
been developed to detect amebic antigen in stool. Available antigen detection
76
methods vary in their sensitivity and specificity and many cannot reliably
77
distinguish between E. histolytica and E. dispar.
78
The E. HISTOLYTICA II ELISA (TECHLAB, Blacksburg VA) uses monoclonal
79
antibodies against the E. histolytica Gal/GalNAc-specific lectin. It is the only FDA
3
80
approved commercially available microplate ELISA known to specifically detect E.
81
histolytica and exclude infection by other Entamoeba species (19). This ELISA
82
exhibits greater sensitivity than the combination of microscopy and culture but has
83
been reported to exhibit lower sensitivity (79%) and specificity (96%) relative to
84
real-time PCR (20). The remel ProSpecT™ E. histolytica microplate assay (THERMO
85
FISCHER, USA) also exhibits greater sensitivity than microscopy and culture, but it is
86
known to cross-react to some extent with E. dispar.
87
Here we report a multi-site comparative analysis of the third generation E.
88
HISTOLYTICA QUIK CHEK™ with the E. HISTOLYTICA II ELISA and ProSpecT™ E.
89
histolytica microplate assay. We demonstrate in stool samples from Bangladesh and
90
South Africa that this rapid immunochromatographic cassette exhibits robust
91
detection of E. histolytica relative to the existing microplate assays. The decreased
92
turn around time, simple platform, and easy interpretation of rapid tests such as the
93
QUIK CHEK offer significant advantages in clinical settings, allowing a health care
94
provider to provide point-of-care diagnosis and treatment.
95
Methods
96
Study populations and clinical samples
97
Frozen clinical stool specimens from South Africa were collected for a study
98
of opportunistic infections among patients at risk of HIV infection in Limpopo
99
province. Testing was authorized by the Health, Research and Ethical committee of
100
the University of Venda. Clinical diarrheal and surveillance stool specimens from
101
Dhaka, Bangladesh were obtained in a prospective study of infants and children
102
living in a slum community. 310 samples were obtained from the ICDDR,B sample
103
repository and 148 from the University of Venda. Testing was first authorized by the
104
Research Review and Ethical Review Committees of the International Center for
105
Diarrheal Disease Research, Bangladesh (ICDDR,B) and the Institutional Review
106
Board of the University of Virginia. 1 sample from the ICDDR,B and 8 samples from
107
South Africa had insufficient material for testing. 160 of the samples from the
108
ICDDR,B population were tested at TECHLAB. All remaining samples with sufficient 4
109
material for all three tests were tested at the ICDDR,B. Frozen un-fixed stool
110
samples were shipped on dry ice to maintain a cold chain in transit. As part of the
111
sample intake procedure, fresh unfrozen stool samples were assessed by
112
microscopy for protozoan parasites. These archival data were used in part to enrich
113
our sample pool in samples likely to contain Entamoeba species.
114
Each specimen was tested by E. HISTOLYTICA QUIK CHEK™ (TECHLAB,
115
Blacksburg, Virginia), E. HISTOLYTICA II ELISA (TECHLAB, Blacksburg, Virginia), and
116
Remel ProSpecT ™ ELISA (THERMO FISCHER, USA). Discrepant results were tested
117
by real-time PCR for E. histolytica and E. moshkovskii and a nested PCR for E. dispar.
118
Detection of E. histolytica by E. HISTOLYTICA QUIK CHEK™
119
All reagents and specimens were brought to room temperature prior to
120
testing. 25 l of liquid stool or a small 2 mm diameter portion of solid stool was
121
homogenized with 500 l of diluent premixed with 1 drop of conjugate. External
122
controls were prepared for each set of samples by adding one drop of positive
123
control or 25 l diluent to 500 l diluent. 500 l of sample or control was added to
124
the sample well of a membrane device and incubated for 15 minutes. 300 l of wash
125
buffer followed by 2 drops of substrate were added directly to the reaction window.
126
Internal control (C) and test (T) lines were read after 10 minutes. Figure 1 depicts
127
examples of negative and positive results on the QUIK CHEK.
128
Detection of E. histolytica by E. HISTOLYTICA II and ProSpecT™ microwell
129
ELISAs
130
Frozen liquid and solid stool specimens were handled and shipped according
131
to the kit manufacturers’ instructions. Testing was performed according to the
132
instructions of the manufacturer for both ELISA kits. Interpretation for both assays
133
was based on a single OD reading at 450 nm with negative control subtracted OD
134
values greater than or equal to 0.050 considered positive for E. histolytica.
5
135
Resolution of discrepant results by Entamoeba species-specific polymerase
136
chain reaction
137
Samples that were discrepant by at least one of the three antigen detection
138
methods were tested by PCR for E. histolytica, E. dispar, and E. moshkovskii. The E.
139
histolytica real-time PCR assay was performed as part of a multiplex assay including
140
Giardia intestinalis and Cryptosporidium spp.. according to the protocol described by
141
Haque et al. (17). A 2-step nested PCR was performed to detect E. dispar according
142
to the protocol described by Haque et al. (18). An in-house TaqMan assay was used
143
to detect E. moshkovskii (Em-f GCG CAA GCT AAG TTT CTA GGA ATG AGG; Em-r TCC
144
TTT TAA TCC TTC TCT CGA AAT GTC CGAA; Em-P HEX-CGA AGG AGA TGA AGT GAG
145
TAA TCA CTT TATC –BHQ1). Reaction conditions were briefly as follows: 2 mmol/L
146
MgCl2, 0.4 mol/L each of forward and reverse primers, 0.08 mol/L of probe, 3 L
147
of extracted DNA, and Qiagen master mix with a final reaction volume of 25 l. The
148
cycling conditions consisted of 15 minutes at 95°C followed by 40 cycles of 15
149
seconds at 95°C and 1 minute at 58°C.
150
Results
151
458 samples were collected for the study. 449 samples had sufficient
152
material and were tested by the E. HISTOLYTICA QUIK CHEK™, E. HISTOLYTICA II
153
ELISA, and ProSpecT™ Entamoeba histolytica microplate assay. 9 samples were
154
excluded from the analyses because of insufficient material. Age and gender data
155
were available for 350 and 424 samples respectively. 87% of the samples came from
156
participants less than 18 years of age. On average, the samples were derived from
157
populations that were 46% male and 54% female.
158
Comparative analysis of the E. HISTOLYTICA QUIK CHEK™ assay for rapid
159
detection of E. histolytica in stool.
160
84 samples were positive and 327 were negative by all three detection
161
methods. 3 samples positive by QUIK CHEK and 3 samples negative by QUIK CHEK
162
were discrepant with the E. HISTOLYTICA II ELISA. While 10 samples positive and
6
163
24 samples negative by QUIK CHEK were discrepant with the ProSpecT™ E.
164
histolytica ELISA. These 40 instances of discrepancy were attributable to 38 child
165
stool samples. 2 samples were discrepant in both analyses. The 38 samples
166
discrepant in either of the two comparative analyses were subsequently resolved by
167
PCR for E. histolytica, E. dispar, and E. moshkovskii.
168
Table 1 consists of two 2 x 2 tables comparing the E. HISTOLYTICA QUIK
169
CHEK™ to the E. HISTOLYTICA II ELISA (a) and the ProSpecT™ E. histolytica ELISA
170
(b). The E. HISTOLYTICA
171
99.4%) sensitivity and 99.2 percent (95% CI: 97.5%-99.8%) specificity compared
172
with the E. HISTOLYTICA II ELISA. The QUIK CHEK exhibited 78.4 percent (95% CI:
173
69.6%-85.6%) sensitivity and 97.0 percent (95% CI 94.6%-98.6%) specificity
174
compared with the ProSpecT™ E. histolytica ELISA. Overall, the E. HISTOLYTICA QUIK
175
CHEK™ exhibited the strongest correlation with the E. HISTOLYTICA II ELISA results
176
prior to resolution by species specific PCR.
177
Resolution of discrepant results by Entamoeba species specific polymerase
178
chain reaction
QUIK CHEK demonstrated 96.9 percent (95% CI: 91.2%-
179
Table 2 consists of two 2 x 2 tables comparing the E. HISTOLYTICA QUIK
180
CHEK™ to the E. HISTOLYTICA II ELISA with discrepant results resolved by PCR for
181
E. histolytica (a) and to the ProSpecT™ E. histolytica ELISA with discrepant results
182
resolved by PCR for E. histolytica (b). 38 samples were discrepant by one of the
183
three antigen detection tests. After PCR resolution of discrepant results between the
184
two assays, the E. HISTOLYTICA QUIK CHEK™ exhibited sensitivity and specificity
185
compared to the E. HISTOLYTICA II ELISA of 98.0 percent (95% CI: 92.9%-99.8%)
186
and 100 percent (95% CI: 99.0%-100%) respectively. The same discrepant
187
resolution yielded sensitivity and specificity values of 97.0 percent (95% CI: 91.5%-
188
99.4%) and 100 percent (95% CI: 99.0%-100%) for the E. HISTOLYTICA QUIK
189
CHEK™ compared to the ProspecT™ E. histolytica ELISA.
190
In order to identify possible sources of amebic cross reactivity, samples with
191
discrepant results were also analyzed by nested PCR for E. dispar and real-time PCR 7
192
for E. moshkovskii. No samples tested positive by PCR for E. moshkovskii. 13 samples
193
tested positive for E. dispar. These E. dispar positive samples all tested positive for E.
194
histolytica by ProSpecT™ ELISA and negative for E. histolytica by the E. HISTOLYTICA
195
QUIK CHEK™, E. HISTOLYTICA II ELISA and E. histolytica-specific PCR. All other
196
discrepant samples tested negative for E. dispar. These data suggest that the
197
ProSpecT™ ELISA may cross-react to some degree with E. dispar.
198
In a supplementary analysis, we tested 9 additional clinical stool samples
199
from Bangladesh, which were screened by PCR for single Entamoeba species. 3
200
samples were positive for E. histolytica and negative for E. dispar and E. moshkovskii.
201
3 samples were positive for E. dispar and negative for E. histolytica and E.
202
moshkovskii. And 3 samples were positive for E. moshkovskii and negative for E.
203
histolytica and E. dispar. The 3 E. histolytica positive samples tested positive by E.
204
HISTOLYTICA QUIK CHEK™. E. dispar and E. moshkovskii positive samples did not
205
cross-react with the E. HISTOLYTICA QUIK CHEK™ (Data not shown). These
206
preliminary data suggest that the E. HISTOLYTICA QUIK CHEK™ is unlikely to cross-
207
react with E. dispar or E. moshkovskii.
208
Discussion
209
The major finding of our multi-site clinical evaluation of the E. HISTOLYTICA
210
QUIK CHEK™ is that this rapid detection method demonstrates comparable
211
performance with two ELISA-based E. histolytica antigen detection methods.
212
Furthermore, we demonstrate through PCR analysis of discrepant results, a high
213
rate of E. histolytica false positivity for the ProSpecT ™ E. histolytica ELISA relative to
214
the other methods used. This observation may in part be explained by cross
215
reactivity of this test with nonpathogenic E. dispar, which is supported by the
216
presence of E. dispar DNA in more than half of the samples called positive for E.
217
histolytica by the ProSpecT™ ELISA and negative by other antigen detection
218
methods and PCR.
219
A previous study by Korpe et al., reported that the E. HISTOLYTICA QUIK
220
CHEK™ exhibited 100% sensitivity and specificity relative to the E. HISTOLYTICA II 8
221
ELISA (21). While our results are consistent with these findings, we expanded our
222
analysis to include comparison of the QUIK CHEK with the ProSpecT™ E. histolytica
223
ELISA and clinical stool specimens from populations in both Bangladesh and South
224
Africa.
225
Our results support the finding that the rapid E. HISTOLYTICA QUIK CHEK™
226
compares well for detection of E. histolytica in stool with the E. HISTOLYTICA II
227
ELISA. The majority of discrepant results in our study were attributable to the
228
ProSpecT™ E. histolytica ELISA. Using E. histolytica specific real-time PCR, we
229
demonstrated that 21 out of 38 discrepant samples (55.3%) were the result of
230
ProSpecT™ false positivity; 10 out of 38 (26.3%) resulted from ProSpecT™ false
231
negativity; while only 3 out of 38 (7.9%) resulted from QUIK CHEK false negativity; 4
232
out of 38 (10.5%) resulted from E. HISTOLYTICA II ELISA false negativity; and 1 out
233
of 38 (2.6%) resulted from E. HISTOLYTICA II ELISA false positivity. This analysis
234
was limited to the discrepant samples tested by PCR. We detected E. dispar in 13 out
235
of the 21 samples (61.9%) that were false positive by ProSpecT™ ELISA. When
236
compared to the E. HISTOLYTICA II ELISA, which is recommended by the Centers for
237
Disease Control (CDC) for specific diagnosis of E. histolytica infection, our results
238
demonstrate that the QUIK CHEK is a robust point-of-care assay to specifically
239
identify E. histolytica in stool specimens.
240
Our use of real-time PCR in this study was limited to discrepant resolution.
241
Future studies may seek to rigorously compare this rapid test with real-time PCR to
242
assess its true limits of detection. We also limited our species coverage in this study
243
to E. histolytica, E. dispar, and E. moshkovskii. While cross-reactivity with E. dispar is
244
likely to be the greatest concern in clinics and epidemiological studies, it will be
245
interesting in future studies to assess the reactivity of this assay with a wider range
246
Entamoeba species and perhaps other fecal microbes.
247 248
Acknowledgments
9
249
This work was supported by NIH 5R01 AI043596-16 and a Fulbright
250
research grant to HPV. The authors acknowledge that Joel Herbein and Blake
251
Hanbury are employed by TECHLAB and were involved in the development of both
252
the E. HISTOLYTICA QUIK CHEK™ assay and the E. histolytica II ELISA evaluated in
253
this manuscript. William A. Petri Jr. receives licensing fees from TECHLAB. These
254
fees are donated in full to the American Society for Tropical Medicine and Hygiene
255
(ASTMH).
256
References
257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287
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homosexual men: further evidence for the occurrence of pathogenic strains. Trans. R. Soc. Trop. Med. Hyg. 84:250–251. 10. Gunther J, Shafir S, Bristow B, Sorvillo F. 2011. Amebiasis-Related Mortality among United States Residents, 1990–2007. The American Journal of Tropical Medicine and Hygiene 85:1038 –1040. 11. Lim P-L, Han P, Chen LH, MacDonald S, Pandey P, Hale D, Schlagenhauf P, Loutan L, Wilder-Smith A, Davis XM, Freedman DO, GeoSentinel Surveillance Network. 2012. Expatriates ill after travel: results from the Geosentinel Surveillance Network. BMC Infect. Dis. 12:386. 12. Dolabella SS, Serrano-Luna J, Navarro-García F, Cerritos R, Ximénez C, Galván-Moroyoqui JM, Silva EF, Tsutsumi V, Shibayama M. 2012. Amoebic liver abscess production by Entamoeba dispar. Ann Hepatol 11:107– 117. 13. Ximénez C, Cerritos R, Rojas L, Dolabella S, Morán P, Shibayama M, González E, Valadez A, Hernández E, Valenzuela O, Limón A, Partida O, Silva EF. 2010. Human Amebiasis: Breaking the Paradigm? Int J Environ Res Public Health 7:1105–1120. 14. Guzmán-Silva MA, Santos HLC, Peralta RS, Peralta JM, De Macedo HW. 2013. Experimental amoebic liver abscess in hamsters caused by trophozoites of a Brazilian strain of Entamoeba dispar. Exp. Parasitol. 134:39– 47. 15. Shimokawa C, Kabir M, Taniuchi M, Mondal D, Kobayashi S, Ali IKM, Sobuz SU, Senba M, Houpt E, Haque R, Petri WA Jr, Hamano S. 2012. Entamoeba moshkovskii is associated with diarrhea in infants and causes diarrhea and colitis in mice. J. Infect. Dis. 206:744–751. 16. Royer TL, Gilchrist C, Kabir M, Arju T, Ralston KS, Haque R, Clark CG, Petri WA Jr. 2012. Entamoeba bangladeshi nov. sp., Bangladesh. Emerging Infect. Dis. 18:1543–1545. 17. Haque R, Roy S, Siddique A, Mondal U, Rahman SMM, Mondal D, Houpt E, Petri WA Jr. 2007. Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. Am. J. Trop. Med. Hyg. 76:713–717. 18. Haque R, Ali IKM, Akther S, Petri WA. 1998. Comparison of PCR, Isoenzyme Analysis, and Antigen Detection for Diagnosis of Entamoeba histolytica Infection. J Clin Microbiol 36:449–452. 19. Haque R, Mollah NU, Ali IKM, Alam K, Eubanks A, Lyerly D, Petri WA. 2000. Diagnosis of Amebic Liver Abscess and Intestinal Infection with the TechLab Entamoeba histolytica II Antigen Detection and Antibody Tests. J Clin Microbiol 38:3235–3239. 20. Roy S, Kabir M, Mondal D, Ali IKM, Petri WA, Haque R. 2005. Realtime-PCR assay for diagnosis of Entamoeba histolytica infection. J. Clin. Microbiol. 43:2168–2172. 21. Korpe PS, Stott BR, Nazib F, Kabir M, Haque R, Herbein JF, Petri WA. 2012. Evaluation of a Rapid Point-of-Care Fecal Antigen Detection Test for Entamoeba histolytica. Am J Trop Med Hyg 86:980–981.
11
334 335 336 337 338 339 340 341 342 343
Tables and Figure legends
344 345
Figure 1. E. HISTOLYTICA QUIK CHEK™ negative and positive results. Left: a
346
negative result on the QUIK CHEK. Right: a positive result. C = control line (positive
347
internal control), T = test line (sample readout).
348 349 350 351 352 353 354 355 12
356 357 358 359 360 361 362 363 364 E. HISTOLYTICA II ELISA
a.
E. HISTOLYTICA QUIK CHEK™
Positive
Negative
Total
Positive
94
3
97
Negative
3
349
352
Total
97
352
449
QUIK CHEK™
Sensitivity 96.9 Specificity 99.2
95% CI 91.2 - 99.4
97.5 - 99.8
ProSpecT™E. histolytica ELISA
b.
E. HISTOLYTICA
%
Positive
Negative
Total
Positive
87
10
97
Negative
24
328
352
Total
111
338
449
% Sensitivity 78.4 Specificity 97.0
95% CI 69.7 - 85.6
94.6 - 98.6
13
365 366
Table 1. Comparative analysis of the E. HISTOLYTICA QUIK CHEK™ assay for
367
rapid detection of Entamoeba histolytica in stool. 449 frozen clinical diarrheal
368
and surveillance stool specimens were assayed to compare the performance of the
369
recently released E. HISTOLYTICA QUIK CHEK™ with those of two commercially
370
available microplate based methods for detection of E. histolytica in stool. Positive
371
and negative predictive agreements with each assay are reported with 95%
372
confidence intervals. (a) The QUIK CHEK exhibited 96.9% and 99.2% positive and
373
negative predictive agreement with the E. HISTOLYTICA II ELISA (TECHLAB®)
374
respectively. (b) The QUIK CHEK exhibited 78.4% and 97.0% positive and negative
375
predictive agreement with the ProSpecT™ E. histolytica ELISA (Remel).
376 E. HISTOLYTICA II ELISA or PCR
a.
E. HISTOLYTICA QUIK CHEK™
Positive
Negative
Total
Positive
97
0
97
Negative
2
350
352
Total
99
350
449
QUIK CHEK™
Sensitivity 98.0
95% CI 92.9 - 99.75
Specificity 100
99.0 - 100
ProSpecT™E. histolytica ELISA or PCR
b.
E. HISTOLYTICA
%
Positive
Negative
Total
Positive
97
0
97
Negative
3
349
352
Total
100
349
449
% Sensitivity 97.0 Specificity 100
95% CI 91.5 - 99.4
99.0 - 100
14
377 378
Table 2. Comparison of the E. HISTOLYTICA QUIK CHEK™ with two microwell
379
assays after real-time PCR resolution of discrepant results.
380
38 samples out of 449 tested resulted in 40 discrepancies when the E. HISTOLYTICA
381
QUIK CHEK™ was compared with two microwell ELISAs. All 38 samples were
382
assayed by the gold standard of detection, real-time PCR for E. histolytica. Sensitivity
383
and specificity compared with each assay after PCR resolution are reported with
384
95% confidence intervals. (a) The E. HISTOLYTICA QUIK CHEK ™ exhibited 98.0%
385
and 100% positive and negative predictive agreement with the E. HISTOLYTICA II
386
ELISA respectively. (b) The E. HISTOLYTICA QUIK CHEK™ exhibited 97.0% and
387
100% sensitivity and specificity compared with the Remel ProSpecT™ E. histolytica
388
ELISA.
15
