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TITLE PAGE. 1
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Title: 3
Early detection of treatment failure in human T. solium taeniasis by coproantigen 4
ELISA detection 5
T. solium taeniasis coproantigen detection is an early indicator of treatment failure 6
for taeniasis 7
8
Running title: 9
Early detection of treatment failure in taeniasis 10
11
Authors: 12
Javier A. Bustos,1,2 Silvia Rodriguez,1,2 Juan A. Jiménez,1,2 Luz M. Moyano,1 13
Yesenia Castillo,1 Viterbo Ayvar,1 James C. Allan,3 Philip S. Craig,4 Armando E. 14
Gonzalez,5 Robert H. Gilman,6 Victor C.W. Tsang,7 and Hector H. Garcia,(*) 1,2 for 15
the Cysticercosis Working Group in Peru 16
17
This study was carried out at: 18
Department of Microbiology and Center for Global Health - Tumbes, 19
Universidad Peruana Cayetano Heredia, Lima, Peru 20
Av. Honorio Delgado 430, Urb. Ingeniería, S.M.P. Lima 31- Perú. 21
Telephone: Int+ 511 3287360 22
23
24
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05428-11 CVI Accepts, published online ahead of print on 15 February 2012
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Author´s Affiliation: 25
1 Department of Microbiology and Center for Global Health - Tumbes, Universidad 26
Peruana Cayetano Heredia, Lima, Peru 27
2 Cysticercosis Unit, National Institute of Neurological Sciences, Lima, Peru 28
3 Pfizer Inc, Madison, New Jersey 07940, USA 29
4 Cestode Zoonoses Research Group, University of Salford, Salford, England, UK 30
5 School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, 31
Lima, Peru. 32
6 Department of International Health, Johns Hopkins Bloomberg School of Public 33
Health, Baltimore, MD, USA 34
7 Department of Biology, Georgia State University, Atlanta, Georgia 35
36
(*) Corresponding author: 37
Hector H. Garcia, MD, PhD. 38
Department of Microbiology, Universidad Peruana Cayetano Heredia. H. Delgado 39
430, SMP, Lima 31, Perú. 40
Telephone: Int+ 511 3287360, Fax: Int+ 511 3287382 41
e-mail address: [email protected] 42
43
Word count: Abstract 252 44
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1. SUMMARY. 46
47
Taenia solium causes taeniasis and cysticercosis, a zoonotic complex associated 48
with a significant burden of epilepsy in most countries. Reliable diagnosis and 49
efficacious treatment of taeniasis are needed for disease control. Currently, cure 50
can only be confirmed after a period of at least one month, by negative stool 51
microscopy. This study assessed the performance of coproantigen ELISA (CoAg-52
ELISA) detection for the early evaluation of the efficacy of antiparasitic treatment in 53
human T. solium taeniasis. We followed 69 tapeworm carriers who received 54
niclosamide as standard treatment. Stool samples were collected on days 1, 3, 7, 55
15, 30, and 90 after treatment and processed by microscopy and CoAg-ELISA. The 56
efficacy of niclosamide was 77.9% (53/68). Thirteen patients received a second 57
course of treatment and completed the follow up. CoAg-ELISA was therefore 58
evaluated in a total of 81 cases (68 treatments, 13 re-treatments). In successful 59
treatments (n=64), the proportion of patients with a who became negative CoAg-60
ELISA was 70.2% 62.5% after 3 days, 92.6 89.1% after 7 days, 88.7 96.9% after 15 61
days, and 96.9 100% after 30 days. In treatment failures (n=17), the CoAg-ELISA 62
was positive in 85.7 70.6% of patients after three days, 94.1% after 7 days, and in 63
100% after 7, 15 and 30 days. Only 2 of 17 treatment failures became positive for 64
microscopy by day 30. The presence of one scolex but not multiple scolices in post-65
treatment stools was strongly associated with cure (OR 52.5% p<0.001). CoAg-66
ELISA is useful to assess treatment failure in taeniasis. Early assessment at day 7 67
15 would detect treatment failure before patients become infective. 68
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2. INTRODUCTION. 70
Neurocysticercosis (NCC) is the most frequent cause of late-onset epilepsy in 71
the world and a growing public health problem in developed countries.(8,9,12,30) 72
This zoonotic cestode has a complex two-host life cycle. Humans are the only 73
definitive host of the adult tapeworm. However, both humans and pigs may harbor 74
the larval stage (cysticerci) in their tissues.(10) The tapeworm is composed of a 75
scolex or head, its neck and a series of proglottids (immature, mature, and gravid as 76
they are separated from the scolex by new proglottids). Each gravid proglottid 77
contains around 50,000 eggs, which are intermittently removed and released into the 78
environment with the feces. Humans and pigs are infected by ingestion of Taenia 79
eggs by the fecal-oral route. The embryos contained in the eggs are released, cross 80
the intestinal mucosa and are then dispersed throughout the body by the circulatory 81
system. Humans complete the cycle when they consume poorly cooked pork 82
infected with cysticerci which then develops into an intestinal tapeworm.(33) 83
84
The standard diagnostic tool for taeniasis is stool microscopy to detect Taenia 85
sp. eggs after concentration. Its specificity is high with a trained operator, but its 86
sensitivity is low because of the variable numbers of eggs excreted in stools and the 87
small volume of stools which are examined.(10) Also, microscopy cannot 88
discriminate between T. solium and T. saginata. Detection of specific tapeworm 89
antigens in stools using ELISA (CoAg-ELISA) has a better diagnostic sensitivity. 90
The CoAg-ELISA for T. solium was developed by Allan and collaborators and is 91
currently the most reliable test for the diagnosis of taeniasis. This ELISA test is 92
performed using hyperimmune rabbit sera raised against somatic antigens of T. 93
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solium adult worm.(1) In epidemiological settings, this assay detects around 2.5 94
times more cases of taeniasis than microscopy does.(3,11) Somatic antigens are 95
released even at immature, pre-patent tapeworm stages and can be detected from 96
the first week after infection as demonstrated in animal models.(6) This technique 97
was 98% sensitive and 99% specific when evaluated by Zamora et al in 2004 using 98
42 known positive stool samples and 163 controls (75 stool samples from patients 99
from a non-endemic area of Peru, and 88 stool samples from US volunteers).(34) 100
Serum stage-specific antibodies against the adult tapeworm can also be detected by 101
immunoblot or ELISA. These assays seem highly sensitive and specific, however 102
their positive predictive value depend on the survival span of circulating antibodies 103
about which much remains unknown.(15,16,21,32) 104
105
Taeniasis/cysticercosis is an eradicable zoonotic disease of which the 106
tapeworm carrier is the only source of infection, thus the effective treatment of 107
taeniasis is a key an important intervention in clinical settings, as well as for control 108
programs.(29) Currently, niclosamide is considered the treatment of choice and its 109
efficacy is claimed to be over 90%.(4,5,7) However, post treatment evaluations have 110
been based on microscopy, likely over-estimating the efficacy of niclosamide. This 111
study was designed to assess the ability of CoAg-ELISA for early detection of 112
treatment success or failure in T. solium tapeworm carriers. 113
114
115
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3. MATERIALS AND METHODS. 117
Study design. Prospective study in recently treated T. solium tapeworm carriers to 118
assess the performance of CoAg-ELISA to define the efficacy of treatment on days 119
3, 7, 15, and 30 post treatment. 120
121
Study population and settings. This study was performed at the Center for Global 122
Health of the Universidad Peruana Cayetano Heredia in Tumbes, Northern Coast of 123
Peru.(24) The study included consecutive patients with confirmed taeniasis due to 124
T. solium who received antiparasitic treatment with niclosamide(7,18) and who were 125
later confirmed by the recovery of parasitological material (gravid proglottids or 126
scolex) for histological species definition and/or confirmation by polymerase chain 127
reaction, followed by restriction enzyme analysis.(23) Participants were asked to 128
collect all immediate post treatment stools and to collect follow up stool samples on 129
days 1, 3, 7, 15 and 30 and 90 post-treatment. These patients had been diagnosed 130
in epidemiological studies by our group in the Tumbes area and prescribed 131
treatment as per standard of care (oral niclosamide in a single dose of 2 g in adults, 132
1.5 g in children over 35 kg and 1 g in children of 11-34 kg). All patients were asked 133
to consume a low fiber diet three days before antiparasitic treatment and were 134
purged with Nulitely® (105 g/L polyethylene glycol 3350, 1.43 g/L sodium 135
bicarbonate, 2.8 g/L sodium chloride, 0.37 g/L potassium chloride) two hours before 136
and after treatment.(7,18) The study and consent forms were reviewed and 137
approved by the Institutional Review Board of the Universidad Peruana Cayetano 138
Heredia. 139
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Study intervention. All excreted stool samples during the day of treatment were 141
collected and a macroscopic search for proglottids and scolices was performed. 142
Follow-up stool samples were also collected on days 1, 3, 7, 15, 30, and 90 after 143
treatment and processed for both microscopy and CoAg-ELISA. Successful 144
treatment was defined as negative results for egg detection by microscopy and 145
negative results for CoAg-ELISA at day 90 or after. A treatment failure was defined 146
as someone with a positive stool microscopy result or in two consecutive CoAg-147
ELISA positive stool samples at day 30 post-treatment or after. Non-cured patients 148
received a second course of niclosamide and were followed in a similar manner for 149
an additional 90 days period. 150
151
Preparation and analysis of fecal samples. Stool samples were collected and 152
preserved by homogenization in phosphate buffer saline 5% formaldehyde (1:4), 153
shook and left to sediment. Twenty milliliters of the homogenized sample were 154
processed by stool microscopy after concentration (tube sedimentation).(25,31) 155
156
Coproantigen ELISA detection. Aliquots of 1.5 ml of the stool supernatant were 157
used for CoAg-ELISA after centrifugation at room temperature at 3,200g for 10’. 158
The CoAg-ELISA technique was performed essentially as described by Allan et al, 159
using hyperimmune rabbit anti-T. solium IgG as capture antibody and goat anti T. 160
solium peroxidase-labeled IgG as conjugate.(1,3) The performance of this 161
technique had been previously evaluated using a battery of 42 stool samples from 162
patients with parasitologically confirmed T. solium cases, 75 stool samples from 163
patients from a non-endemic area of Peru, and 88 stool samples from US 164
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volunteers, with values of 98% for sensitivity (41/42, 95%CI 93-100), and 99% for 165
specificity (161/163, 95%CI 97-100).(34) Processed samples were read with a 166
spectrophotometer (Molecular Devices Inc, CA, USA) at 650 nm. Using a known 167
positive pool (P1) we calculated a percent of positivity (PP) as OD of the sample / 168
(OD P1)*100, to increase the comparability of the results between plates. A cut off 169
was determined using a ROC curve. 170
171
Data analysis. Positive predictive values of CoAg-ELISA for treatment failure and 172
negative predictive values of CoAg ELISA for cure at days 3, 7, 15 and 30 after 173
niclosamide treatment were extracted from 2x2 contingency tables comparing the 174
CoAg result with the treatment outcome (cure or failure) at each time point. To 175
progression of the proportions of CoAgELISA positive results in treatment failures 176
and negative CaAgELISA results in cured patients were calculated as cumlative 177
proportions at each time point. A similar analysis was done for microscopy. Finding 178
the tapeworm scolex in the immediate post-treatment stool sample was also 179
evaluated as predictor of efficacy. Data analysis was performed on STATA 180
software (StataCorp, College Station, TX). 181
182
183
4. RESULTS 184
Between 2004 and 2007, parasitic material was recovered and allowed species 185
definition in 86 individuals with a presumptive diagnosis of taeniasis who had 186
received antiparasitic treatment followed by a purgative as per standard of care. 187
Sixty-nine of them were confirmed as T. solium tapeworm carriers, 16 were 188
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diagnosed as T. saginata carriers and in one case the characterization was 189
inconclusive. The 69 patients with T. solium infection were mostly women (48, 190
69.6%) and had a mean age of 33 years (SD 15.7 years). Follow up of the initial 191
treatment was completed in 68 out of 69 patients. Treatment efficacy in this 192
population was 77.9% (53/68, 95% CI 66.7 – 86.2). All 15 individuals with 193
treatment failure had a positive CoAg-ELISA result and all but one had 194
parasitological diagnosis (finding of Taenia eggs [n=14], proglottids [n=10] or 195
scolex [n=9]) as a confirmation of failure at days 30 or 90 or during re-treatment. 196
The individual without parasitological confirmation of treatment failure had 197
consistently increasing CoAg-ELISA values at days 3, 7, 15 and 30 and a decision 198
of re-treatment was taken, becoming CoAg-ELISA negative after re-treatment. 199
Thirteen of the 15 non-cured patients received a second course of niclosamide and 200
were followed-up in a similar manner; 11 out of 13 were cured after the second 201
treatment. Failure after a second treatment was demonstrated in one case by a 202
gradual increase in coproantigen levels (up to eight times in a logarithmic scale) 203
after an initial drop to borderline immediately after treatment, and by expulsion of 204
more parasitic material after a third re-treatment in the other. Thus, we considered 205
81 courses of treatment and follow-up (68 treatments and 13 re-treatments) to 206
evaluate the performance of CoAg-ELISA and microscopy at days 3, 7, 15, and 30. 207
208
Predictive value Performance of coproantigen ELISA post-treatment follow up. 209
The cumulative proportions of cured and non cured patients and their respective 210
CoAg-ELISA results are shown in Table 1. In every case of treatment failure the 211
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CoAg-ELISA was positive after day 7 15 of follow up. In cured patients, CoAg-212
ELISA results became negative at day 30 in all cases. 213
214
Predictive values of a negative CoAg-ELISA for successful cure were 95.2% 215
(40/42) at day 3, and 100% thereafter (50/50 at day 7, 55/55 at day 15 and 62/62 at 216
day 30). Predictive values of a positive CoAg-ELISA to detect treatment failure were 217
41.4% (12/29) at day 3, 80.0% (16/20) at day 7, 69.6% (16/23) at day 15, and 89.5% 218
(17/19) at one month (Table 2). The increase in predictive value at day 7 compared 219
with day 15 is most likely an artifact due to fewer samples examined on that 220
particular day. 221
222
Stool microscopy post-treatment follow up. The same 81 follow up courses were 223
considered. Stool microscopy was a poor indicator of treatment success even at day 224
30 of follow up. The cumulative proportions of cured and non-cured patients and 225
their respective parasitological results are shown in Table 3. 226
227
Post-treatment recovery of tapeworm scolex. We were able to find at least one 228
tapeworm scolex in the immediate post-treatment stools of 58 of 81 cases (71.6%). 229
In five cases (5/58) more than one scolex were detected. Almost all patients in whom 230
at least one scolex was recovered were cured (56/58, 96.6%, 95% IC 88.3%-231
99.1%), compared with only 34.8% of patients in whom no scolex was found (8/23, 232
95% CI 18.8 – 55.1). It follows that scolex recovery was strongly associated with 233
treatment efficacy (56/58 versus 8/23, OR 52.5, 95% CI 10.1 – 273.6, p<0.001). The 234
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only two treatment failures in this group corresponded to 2 out of the 5 patients in 235
whom more than one scolex was recovered. 236
237
238
5. DISCUSSION. 239
CoAg-ELISA can reliably predict treatment success or failure as soon as 240
seven days after treatment of human taeniasis by Taenia solium. In this series, a 241
negative coproantigen at day seven or after was an absolute indicator of cure, 242
while only 8% of cured patients remained CoAg-ELISA positive at day 7, and less 243
than 4% after one month. The CoAg-ELISA assay can identify treatment failures 244
several weeks before they become infective, as shown in this work confirming very 245
earlier studies using a Hymenolepis diminuta rat model in 1988.(22) Not 246
surprisingly, microscopy was unable to detect treatment failures by day 7 and 15 247
and only detected 2 out of 17 failures by day 30 (11.8%). It is highly likely that a 248
great proportion of the tapeworm strobila is eliminated after treatment and thus it 249
takes weeks or months for the parasite to pass gravid progglotids or eggs again. 250
In de novo infections, the pre-patent period is assumed to be around 3 months. 251
(27, 33) 252
253
A few cured patients were CoAg-ELISA positive at 7, 15 or 30 days after 254
treatment. We cannot rule out that these tapeworms did not die immediately but 255
were only weakened and cured later by natural evolution or by the host’s immunity. 256
This is unlikely, however, since their antigen levels dropped abruptly and stayed at 257
very low levels (albeit positive as defined by the ELISA cut off). Alternatively, some 258
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tapeworm antigens or cell remnants at the gastrointestinal tract may have 259
remained there and been slowly excreted. Positive results at day 15 (n=7) and 30 260
(n=2) correspond to low, border-line false positive results according the calculated 261
cut-off. Taenia sp eggs were found in the stools of two cured patients at day 7 after 262
treatment. This finding is relevant because even when a purge was used, some 263
treated patients can still be a source of infection even days after treatment. This 264
series was composed of patients treated in a health center after parasitological 265
diagnosis and after a previous period of diet, and purge.(18) Our results cannot be 266
extrapolated to mass population-based treatment campaigns with niclosamide, 267
where the likelihood of further infectivity should be higher. 268
269
As previously described by our group,(18) some patients expelled more than 270
one worm scolex. We found four (8.2%) such cases after the first treatment course, 271
somewhat lower than the 20% (4/20) reported by Jeri et al in 2004.(18) Still, 272
multiple T. solium infections are not such a rare event. In this series they were less 273
prone to cure, and were all women. As expected, scolex elimination is strongly 274
associated to cure, except in the case of individuals with multiple tapeworms.(10) 275
276
A comparison, untreated group was not considered because of ethical 277
reasons, and thus we cannot rule out the chances that some parasites died by 278
natural evolution instead of the antiparasitic effect of niclosamide, or that other 279
patients were re-infected. None of these scenarios seem likely. Whether re-280
infection is possible in human T. solium is unknown and has not been reported. 281
The life span of T. solium tapeworms was claimed to be decades, but later 282
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epidemiological analysis suggested a few years. (2,17,19) Thus, considering a life 283
span of three years, natural death of well-established tapeworms in a two-week 284
period could not account for more than 1 of the 75 (1.3%) tapeworms. Natural 285
death of well established tapeworms in a two-week period could not account for 286
more than 1 of each 75 tapeworms if the life span is three years. 287
288
This series included a selected subgroup of patients, who were treated after 289
diet and purge, and in whom parasite material was recovered, and thus we cannot 290
extrapolate the efficacy of niclosamide (77.9%) to all tapeworm carrier cases. Some 291
considerations here deserve further elaboration. Most other studies on niclosamide 292
reported higher efficacy (37/43, 86%(14); 678/766, 88.5% [mostly T. saginata];(26) 293
46/47, 98% [T. saginata],(28) and others(5,7)). The efficacy was probably found 294
lower because of the higher sensitivity of CoAg-ELISA as a tool to demonstrate the 295
persistency of parasite. These treatment conditions can hardly been achieved in 296
massive treatment campaigns (which also do not normally achieve 100% coverage), 297
consequently it is likely that its efficacy in community-based treatment would be even 298
lower. A lack of efficacy of 20% or more can seriously affect control performance 299
targets in taeniasis/cysticercosis. In clinical settings, the diagnosis, treatment, and 300
follow-up of T. solium tapeworm carriers is relevant, moreover in patients with 301
neurocysticercosis in whom higher prevalence of taeniasis, related to severity of 302
infection, can be expected. (13) 303
304
The positive predictive value of coproantigen detection was not perfect. If the 305
decision is made to define treatment success at day 7, seven to eight percent of 306
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patients with false positive coproantigen results could end up receiving an additional, 307
unnecessary course of treatment. The risks associated to this re-treatment are 308
minimal however, since the effect of niclosamide is only intraluminal and its adverse 309
events are mild and transient. 310
311
Tapeworm carriers are a source of infection for themselves, their households 312
and their villages.(19,20) The effectiveness of treatment must be maximized and 313
thus a rapid confirmation to cure (or treatment failure) is important. To allow a period 314
of months for detection of treatment failure increases the likelihood of losses to 315
follow up and thus some tapeworm carriers will again become sources of infection. 316
Cure of the tapeworm carriers, the only source of infection, is critical to control this 317
zoonotic disease. The CoAg-ELISA test is a useful technique for early detection of 318
treatment success or failure. Unfortunately there is no commercial source for this 319
test at the present time. This technique must be made available in endemic regions 320
at a low cost, in a robust format to allow its use in field conditions. When available, 321
follow up with CoAg-ELISA should be routinely used to evaluate treatment efficacy. 322
323
324
6. ACKNOWLEDGMENTS. 325
Support from the Bill and Melinda Gates Foundation grants 23981 and 326
33848, and the Fogarty International Center - NIH grant D43 TW001140 are 327
acknowledged. Hector H. Garcia is now a Wellcome Trust Senior International 328
Research Fellow. 329
330
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331
332
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diagnosis of intestinal taenia solium taeniais. Am J Trop Med Hyg 71(4 448
suppl):42 449
450
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Table 1. Cumulative percent of CoAg-ELISA results in the follow up of anti-parasitic 460
treatment of T. solium infection. 461
462
Successful Treatments (n=64) Treatment Failures (n=17)
Days after Tx Negative CoAg
result * 95% CI
Positive CoAg
result * 95% CI
3 62.5% (40/64) 50.2 – 73.3 70.6% (12/17) 60.1 – 96.0
7 89.1% (57/64) 79.1 – 94.5 94.1% (16/17) 72.7 – 98.6
15 96.9% (62/64) 89.3 – 99.0 100% (17/17) 81.5 – 100
30 100% (64/64) 94.5 – 100 100% (17/17) 81.5 – 100
90 100% (64/64) 94.5 – 100 100% (17/17) 81.5 – 100
463
• Not all patients complied with all sample collection times. This analysis loses 464
a total of 12 weak positive results at day 7(1), 15(5), 30(2) and 90(4) in 465
successful treatments and 1 negative result at day 90 in treatment failures. 466
• 467
468
469
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Table 02 470
CoAg-ELISA results at days 3, 7, 15, and 30 after treatment 471
472
CoAg-ELISA
Result
Days after antiparasitc treatment (*)
Day 3 Day 7 Day 15 Day 30
NC C NC C NC C NC C
Positive 12 17 16 4 16 7 17 2
Negative 2 40 0 50 0 55 0 62
PV of failure 41.4% (12/29) 80% (16/20) 69.6% (16/23) 89.5% (17/19)
PV of cure 95.2% (40/42) 100% (50/50) 100% (55/55) 100% (62/62)
473
NC: non-cured patients, C: cured patients, PV: predictive value 474
* Not all patients complied with all sample collection times. 475
476
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Table 3. Cumulative percent of microscopy (after concentration) results in the follow 477
up of anti-parasitic treatment of T. solium infection 478
Successful Treatments (n=64) Treatment Failures (n=17)
Days after
Treatment
Negative
Microscopy *
95% CI Positive
Microscopy *
95% CI
3 79.7% (51/64) 68.2 – 87.7 11.8% (2/17) ¥ 3.6 – 34.7
7 92.2% (59/64) 83.0 – 96.5 11.8% (2/17) 3.6 – 34.7
15 100% (64/64) 94.5 – 100 11.8% (2/17) 3.6 – 34.7
30 100% (64/64) 94.5 – 100 23.5% (4/17) 9.7 – 47.6
90 100% (64/64) 94.5 – 100 41.2% (7/17) 21.5 – 64.3
479
* Not all patients complied with all sample collection times. This analysis loses a 480
total of 8 negative results at days 7(2), 15(2), 30(2) and 90(2) in treatment failures. 481
¥ Two positive results were found at day 3, these two non-cured patients had 482
negative results at days 3, 7, 15 and 30 and became positive at day 90. 483
484
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