Journal Pre-proof

How long is too long? Application of acetic acid during colposcopy: A prospective
study

Ziad Hilal, MD, Clemens B. Tempfer, MD, Lucie Burgard, MD, Sadia Rehman, MD,
Günther A. Rezniczek, PhD

PII: S0002-9378(20)30059-4
DOI: https://doi.org/10.1016/j.ajog.2020.01.038
Reference: YMOB 13079

To appear in: American Journal of Obstetrics and Gynecology

Received Date: 4 October 2019

Revised Date: 15 January 2020
Accepted Date: 15 January 2020

Please cite this article as: Hilal Z, Tempfer CB, Burgard L, Rehman S, Rezniczek GA, How long is too
long? Application of acetic acid during colposcopy: A prospective study, American Journal of Obstetrics
and Gynecology (2020), doi: https://doi.org/10.1016/j.ajog.2020.01.038.

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 Hilal et al.

1 How long is too long? Application of acetic acid during colposcopy:

2 A prospective study

4 Ziad Hilal, MD,1,2 Clemens B. Tempfer, MD1, Lucie Burgard, MD1, Sadia Rehman

5 MD,1 and Günther A. Rezniczek, PhD1*

7 From the 1Department of Obstetrics and Gynecology, Ruhr-Universität Bochum,

8 Bochum, Germany; and the 2ZydoLab, Dortmund

10

11 The authors report no conflict of interest.

12

13 Funding: None.

14

15

16 *Corresponding author:

17 Günther A. Rezniczek, PhD

18 Department of Obstetrics and Gynecology

19 Ruhr-Universität Bochum – Marien Hospital Herne

20 Hölkeskampring 40, 44625 Herne, Germany

21 Tel: +49 2323 499 1058; Fax: +49 2323 499 1059

22 E-mail: guenther.rezniczek@rub.de

23

24

25 Word counts: 308 (abstract), 2675 (main text).

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26 CONDENSATION

27 The optimal timing for the colposcopic assessment of acetowhite lesions is 1 minute

28 after the application of acetic acid.

29

30 SHORT TITLE

31 Acetic acid test in colposcopy

32

33 AJOG AT A GLANCE

34 A. Why was this study conducted?

35 While applying acetic acid to the cervix followed by colposcopic assessment is the

36 standard test used to detect dysplastic lesions of the cervix, there is no common

37 standard for performing this test.

38 B. What are the key findings?

39 The optimal timing for the colposcopic assessment of acetowhite lesions is 1 minute

40 after the application of acetic acid. Fading of acetowhite lesions is common, starts

41 after 1 minute, and is more pronounced in high-grade than in low-grade squamous

42 intraepithelial lesions.

43 C. What does this study add to what is already known?

44 Using a prospective design, this study clearly defines the optimal time frames for

45 colposcopic assessment after application of acetic acid, allowing colposcopists to

46 minimize the examination’s duration without compromising reliability.

47

48

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49 ABSTRACT

50 BACKGROUND: Applying acetic acid (AA) to the cervix followed by colposcopic

51 assessment with or without colposcopically directed biopsy is the standard test used

52 to detect dysplastic lesions of the cervix. However, there is no evidence-based

53 common standard defining how to exactly perform this test.

54 OBJECTIVE: To prospectively define the optimal timing for the colposcopic

55 assessment of acetowhite lesions.

56 METHODS: Consecutive women referred to our colposcopy unit were recruited.

57 Using a standardized colposcopy protocol, we recorded the most severe colposcopic

58 lesion (MSCL) 1, 3, and 5 minutes after application of AA (primary study end point).

59 The time to first appearance of the MSCL, highest staining intensity, and fading of

60 MSCL were video-documented (secondary study end points, assessed independently

61 by three raters). Results were compared using parametric and nonparametric tests.

62 RESULTS: 300 women were included. After 1 minute, 290/300 (96.7%) of patients

63 were diagnosed with the MSCL. This proportion did not improve after 3 minutes

64 (290/300 [96.7%]) nor after 5 minutes (233/264 [88.3%]). The proportion of minor and

65 major changes continuously declined over time from 142/300 (47.3%; 1 minute) to

66 107/264 (40.5%; 5 minutes) and from 110/300 (36.7%) to 91/264 (34.5%),

67 respectively. The median time until the first appearance of the MSCL was 13.5

68 (interquartile range [IQR] 3 to 27.25) seconds and was significantly lower in HSIL (7

69 [IQR 1 to 20] seconds) compared to LSIL (19 [IQR 9 to 39.5] seconds; p<0.001). We

70 observed fading of acetowhite lesions in 78% of cases, occurring at a median of 191

71 (IQR 120 to 295) seconds after application of AA. Fading started earlier in HSIL

72 compared to LSIL (179.5 [IQR 110 to 253.25] versus 212.5 [IQR 146.5 to 300];

73 p=0.044). Overall, the net difference between colposcopic assessments at 3 minutes

74 versus at 1 minute was one more HSIL and one less LSIL.

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75 CONCLUSION: It is reasonable to conclude that the best time to identify lesions is 1

76 minute after the application of AA. Continued evaluation for up to 3 minutes may be

77 considered reasonable for an optimal HSIL yield. However, fading of acetowhite

78 lesions is common, especially in HSIL, and supports a recommendation of not

79 prolonging colposcopy beyond 3 minutes.

80

81

82 KEY WORDS: acetic acid test; acetowhite lesion; colposcopy; HSIL; LSIL

83

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84 INTRODUCTION

85 Applying acetic acid (AA) to the cervix followed by colposcopic assessment with or

86 without colposcopically directed biopsy is the standard test used to detect dysplastic

87 lesions of the cervix. The AA test is used worldwide and is an integral part of the

88 management of cervical intraepithelial neoplasia. This test is recommended as

89 standard of care by the International Federation for Cervical Pathology and

90 Colposcopy,1 as well as national societies such as the American Society for

91 Colposcopy and Cervical Pathology (ASCCP)2 and the German Society of

92 Colposcopy and Cervical Pathology (AG-CPC).3

93 Surprisingly, recommendations of how exactly to perform AA testing in order to

94 obtain optimal results vary considerably. For example, historical textbooks such as

95 the original description of colposcopy and the AA test by Hinselmann in 1925

96 recommended to apply AA for up to 5 minutes.4 Recent recommendations include

97 application of 5% AA followed by a delay of 1 to 2 minutes before colposcopic

98 assessment,5 3% AA for 30 to 60 seconds before assessment,6 5% AA for 1 minute

99 followed by immediate assessment,7 or application of 5% AA for one minute followed

100 by another minute of waiting.8 In a large randomized screening trial with >130,000

101 participants, 4% AA with assessment 1 minute after AA application was used as a

102 standard,9 based on the WHO International Agency for Research on Cancer’s

103 handbook on cervical cancer screening10 and their own previous trial.11 In many

104 studies, the specifics of AA testing are not defined at all. It is of note that the ASCCP

105 and other national societies do not delineate the details of the AA test in their

106 colposcopy standards.

107 Therefore, based on the data in the literature and based on existing practice

108 guidelines, there is no common standard for AA testing during colposcopy. Current

109 practice of this important and commonly used diagnostic tool is not evidence-based.

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110 This statement is supported by a PubMed literature search (search terms:

111 colposcopy, acetic acid test, acetowhite; search date: 27-09-2019), through which we

112 identified no controlled trial defining the optimal point of time for AA testing during

113 colposcopy. Since the AA test is an integral part of the management of women with

114 cervical dysplasia, there is a medical need to properly define the specifics of this test

115 in a prospective study.

116 In order to address this issue, we designed a prospective study to investigate

117 the AA test in detail in a large patient population. The aim of our study was to

118 properly define the optimal point of time for the colposcopic assessment of

119 acetowhite lesions after AA application. In addition, we wanted to quantify the

120 increases and decreases of acetowhite lesion severity over time and to specify the

121 proportion and timing of fading of acetowhite staining.

122

123 MATERIALS AND METHODS

124 We recruited consecutive women referred to the colposcopy outpatient unit of the

125 Department of Obstetrics and Gynecology of the Ruhr-Universiät Bochum, Germany,

126 or the doctor’s office of the Institute of Cytology and Immune Cytochemistry,

127 Dortmund, Germany, for further assessment of a pathologic Pap smear. After giving

128 informed consent, a detailed personal history was taken and documented. We used a

129 standardized protocol for colposcopy and AA testing and in all women as follows:

130 using a binocular colposcope (Model 1D LED, Leisegang, Berlin, Germany), the

131 native impression of the cervix was recorded with 7.5x and 15x magnification. Then,

132 acetic acid 5% was applied to the cervix with three pushes using a commercially

133 available household spray can (Wilpeg, Großenlüder, Germany), corresponding to a

134 total volume of 3 ml. Then, the colposcopist assessed and classified the most severe

135 colposcopic lesion (MSCL) 1, 3, and 5 minutes after the application of AA. The

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136 primary end point of this study was the proportion of MSCL after 1, 3, and 5 minutes

137 using the International Federation for Cervical Pathology and Colposcopy Rio

138 classification criteria.1 Secondary study endpoints included the time from AA

139 application to the first appearance of the MSCL, the time from AA application to the

140 highest staining intensity of the MSCL, and the time from AA application to the start

141 of fading of acetowhite staining of the MSCL. These were recorded and video-

142 documented for every patient. Of these, 85 videos were selected (with an allocation

143 of 1:2:2 to histological outcome: negative for dysplasia, LSIL, HSIL/carcinoma) for

144 detailed assessment by three colposcopists to further specify the dynamics of

145 acetowhitening. This sample size was calculated based on the experience that

146 acetowhitening of HSIL is faster than of LSIL lesions (assumed on average to be

147 most intense after 60 seconds) and assuming a fixed standard deviation of ±15

148 seconds, so that a difference in time needed to reach maximum intensity of 20%

149 could be detected at an α of 0.05 with a power of at least 90%, and allowing for a

150 dropout/error rate of 15% (video not of suitable quality, wrong group assignment due

151 to clerical errors). G*Power 3.1.9.2 was used for this calculation.12 All three

152 colposcopists, who were blinded to patient information and histological outcome,

153 assessed each video with regard to the exact time (in seconds) until the first and

154 secondary endpoints occurred. Endpoints not occurring before the end of the

155 observation period (300 seconds after application of AA) were censored with a value

156 of 300 seconds. Waterfall plots were used to visualize the time points of fading in

157 low-grade and high-grade lesions as determined by the three raters; each bar

158 represents one observation. To determine proportions of not faded acetowhite

159 lesions, the time points of fading onset determined by the three raters were averaged

160 per video.

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161 All colposcopists were certified specialists with more than 300 annual

162 colposcopies. The study was approved by the Ethics Committee of the Medical

163 Faculty of Ruhr-Universität Bochum, Bochum, Germany (registration number 18-

164 6370, dated August 24, 2018).

165 Assuming a continuous outcome measure such as the time until the

166 appearance of the MSCL, a sample size calculation with the confidence level set at

167 95% and the confidence interval set at 5%, resulted in a sample size of 278 patients

168 for a population size of 1000 women (roughly corresponding to the number of women

169 seen by a colposcopists with 300 cases/year over 3 years). Therefore, we aimed to

170 recruit at least 300 patients, allowing for approximately 10% of dropouts/protocol

171 violation.

172 Study data were collected and managed using REDCap (Research Electronic

173 Data Capture), a secure, web-based application designed to support data capture for

174 research studies.13 After data collection had been concluded, exported data were

175 further processed in Microsoft Excel (Microsoft Inc., Redmond, WA) and prepared for

176 statistical analyses using SigmaPlot 14 (Systat Software Inc., San Jose, CA).

177 Descriptive statistics are reported using means and standard deviations for normally

178 distributed data and medians and interquartile ranges (IQR) for data not meeting this

179 assumption. Accordingly, statistical analysis was performed using parametric (t-test)

180 or nonparametric tests (Mann-Whitney U test for pairwise comparison and ANOVA

181 on ranks for multiple comparisons).. All P values are two-tailed and P<.05 was

182 considered statistically significant.

183

184 RESULTS

185 From September 2018 to May 2019, we prospectively recruited 305 consecutive

186 women from our colposcopy units into this study. Of these, four women were

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187 excluded due to protocol violation. In one case, study documentation was missing.

188 Thus, data from 300 colposcopies were evaluated. Figure 1 gives a detailed account

189 of the patients’ flow through the study and the workflow. Patient characteristics are

190 shown in Table 1. All patients underwent a standardized colposcopy and AA test

191 protocol as delineated in the Materials and Methods section. Table 2 shows the Pap

192 smear results, the colposcopic findings before and after AA testing, and the

193 corresponding histopathological results. Specifically, type I, type II, and type III

194 transformation zones were identified in 195 (65.0%), 58 (19.3%), and 47 (15.7%) of

195 cases. At the end of colposcopy, i.e. 5 minutes after AA application, colposcopic

196 assessment found a normal cervix in 61 (23.1%), minor changes in 107 (40.5%),

197 major changes in 91 (34.5%), and major changes suspicious for invasion in 1 (0.4%)

198 cases, respectively. The colposcopic findings changed over time. When comparing

199 the colposcopic assessments 1 minute, 3 minutes, and 5 minutes after AA

200 application, the proportion of minor changes continuously declined from 142 (47.3%)

201 after 1 minute to 137 (45.7%) after 3 minutes and 107 (40.5%) after 5 minutes. The

202 proportion of major change lesions also declined over time from 110 (36.7%) to 114

203 (38.0%) and to 91 (34.5%), respectively. Of note, this effect was restricted to minor

204 and major changes and was not observed in the categories ,non-specific findings’,

205 ,miscellaneous findings’, and ,other findings’ (as defined by the Rio classification

206 system1).

207 We were interested in defining the optimal time point for the colposcopic

208 assessment after AA application. Thus, we analyzed the proportion of the MSCL at

209 the time points 1, 3, and 5 minutes after AA application (Table 3). We found that after

210 1 minute, 290/300 (96.7%) of patients were diagnosed with the MSCL. This

211 proportion did not improve after 3 minutes (290/300 [96.7%]) nor after 5 minutes

212 (233/264 [88.3%]). Therefore, the optimal time point for the colposcopic assessment

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213 of acetowhite lesions based on our study is 1 minute after the application of AA. An

214 increase of the severity of acetowhite lesions or a change from no acetowhite lesion

215 to acetowhite lesion from 1 minute and 3 minutes was observed in 8/300 (2.7%) of

216 cases (Table 3). Of these 8 cases, changed their colposcopic appearance from

217 negative/unspecific to minor/major change. In other words, a colposcopic

218 assessment at 1 minute would have missed 5 cases because no biopsies would have

219 been performed (1 HSIL, 2 LSIL, 2 with negative histology). A further increase of

220 lesion severity from 3 to 5 minutes was not observed. Conversely, a decrease of

221 lesion severity from 1 minute to 3 minutes was observed in 8/300 (2.7%) cases

222 (colposcopy: 7 cases from minor change to negative, 1 case from major to minor

223 change; histology: 1 HSIL, 3 LSIL, 4 with negative histology). In other words, a

224 colposcopic assessment at 3 minutes (as compared with 1 minute) would have

225 missed 3 cases of LSIL, because these lesions were no longer visible (the HSIL

226 would still have been biopsied because it only changed from major to minor change).

227 Thus, the net difference between colposcopic assessments at 3 minutes versus at 1

228 minute was one more HSIL and one less LSIL in a population of 300 cases.

229 The median time from application of AA until the first appearance of the MSCL

230 was found to be 13.5 (interquartile range [IQR] 3 to 27.25) seconds (Table 4).

231 Interestingly, the median time until the first appearance of the MSCL was significantly

232 lower in HSIL compared to LSIL (7 [IQR 1 to 20] versus 19 [IQR 9 to 39.5]; p<0.001).

233 The median time from the application of AA until the maximum staining intensity of

234 the MSCL was 50.5 (IQR 29 to 75.75) seconds. Again, the median time until

235 maximum staining intensity was significantly lower in HSIL compared to LSIL (46

236 [IQR 32.25 to 70.75] versus 60 [IQR 44.75 to 81.5] seconds; p=0.039). Of note, we

237 observed the phenomenon of fading of acetowhite lesions over time in the majority of

238 cases (78.4%). In the video analysis, the median time from application of AA until the

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239 start of fading of the MSCL was 191 (IQR 120 to 295) seconds. In line with the

240 previous observations, fading started earlier in HSIL compared to LSIL (179.5 [IQR

241 110 to 253.25] versus 212.5 [IQR 146.5 to 300] seconds; p=0.044). Fading was

242 found to be continuously increasing until the end of the recordings, starting around 50

243 seconds after AA (Figure 2).. Although starting significantly earlier in HSIL compared

244 to LSIL (p=0.016), fading affected the majority of HSIL (86.8%) as well as LSIL

245 (72.7%) cases. This finding underscores the necessity of defining the optimal time

246 point for the colposcopic assessment after application of AA in order to avoid missing

247 lesions due to fading. When we looked at the assessments of the three investigators

248 separately, there was a fair congruence with regard to their determinations of when

249 the MSCL became evident, when lesions reached their maximum staining intensity,

250 and when fading started. No statistically significant variability was noted suggesting

251 an acceptable inter-observer variability (Table 4).

252

253 DISCUSSION

254 Principal Findings

255 The optimal timing of the colposcopic AA test is unclear.1–9 In the present study we

256 aimed to define the optimal timing of the colposcopic assessment of acetowhite

257 lesions after AA application. We found that 97% of patients were diagnosed with the

258 MSCL 1 minute after AA application. This proportion did not improve after 3 and 5

259 minutes. Therefore, we conclude that the optimal point of time for the colposcopic

260 assessment of acetowhite lesions is 1 minute after the application of AA.

261

262 Results and Clinical Implications

263 Our study has implications for clinical practice. Identifying the optimal point of time for

264 the colposcopic assessment after AA testing is important and affects how women

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265 with cervical dysplasia or abnormal Pap smears are being managed. An erroneous

266 use of the AA test may lead to the underdiagnoses of cervical dysplasia with the

267 potential of subsequent progress of such unnoticed lesions. Specifically, performing

268 colposcopic assessment after AA application too early might lead to a reduced

269 sensitivity by overlooking lesions not yet visible. Conversely, waiting for too long may

270 lead to overlooking lesions due to fading of acetowhite staining, a phenomenon we

271 observed in 78% of cases. In addition, performing unnecessarily lengthy tests is time

272 consuming and causes patient discomfort. Based on our prospective data collected

273 under standardized conditions, it can now be stated that the optimal balance between

274 quick diagnosis and optimal test yield is 1 minute. This strategy will identify 97% of

275 the MSCL and this percentage cannot be improved by waiting for a longer time. To

276 the contrary, a continuously increasing number of lesions will go unnoticed from 1

277 minute on due to the fading of acetowhite staining, a phenomenon seen in the vast

278 majority of patients.

279 Interestingly, HSIL and LSIL pick up acetowhite staining very quickly. For

280 example, HSIL became first evident after a median of only 7 seconds and LSIL after

281 a median of only 19 seconds. Although the staining process of HSIL and LSIL

282 progressively intensifies after that until reaching a maximum after 50 seconds, this

283 does not change the fact that the MSCL can already be identified seconds after AA

284 application. However, it is reasonable to wait for 1 minute until colposcopic

285 assessment and colposcopically guided biopsy because this time is needed to reach

286 the maximum test yield of 97%. This sensitivity maximum plateaus between 1 and 3

287 minutes and then even declines after that due to the effect of fading. The potential

288 pitfall of waiting too long is underlined by the observation that HSIL fade significantly

289 quicker than LSIL, thus aggravating the clinical relevance of loss of test sensitivity

290 through waiting too long before colposcopy and biopsy. Some colposcopists argue

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291 that a longer waiting time is better in the false belief that more high-grade lesions will

292 become visible the longer the test lasts. We noted in our study that the contrary is

293 true because fading is not a threshold phenomenon but a continuous process already

294 starting 50 seconds after AA application and continuously increasing, especially in

295 HSIL, until it ultimately affects the vast majority of lesions.

296 The results of our study do not imply that colposcopy should be abruptly

297 stopped after 1 minute. They do imply, however, that the best time to identify lesions

298 is 1 minute after the application of AA. Continued evaluation for up to 3 minutes may

299 be considered reasonable for an optimal HSIL yield. However, fading of acetowhite

300 lesions is common, especially in HSIL, and supports a recommendation of not

301 prolonging colposcopy beyond 3 minutes.

302

303 Strengths and Limitations

304 Our study has strengths and limitations. Strengths are the prospective design, the

305 large number of patients, and the standardized technique, which was defined before

306 the start of the trial. In addition, all study procedures were video-documented

307 ensuring that all patients were treated in the same way. Limitations of our study

308 include selection bias because we recruited only women referred to a specialized

309 colposcopy unit. Therefore, the likelihood of finding LSIL/HSIL is increased compared

310 to an unselected group of women in a population-based screening program. An

311 increased likelihood of a positive diagnostic test might inflate the sensitivity of the AA

312 test. However, since procedural aspects of the test and not test accuracy were the

313 primary endpoint of our study, we believe that the possible selection bias does not

314 affect the validity of our results. Further, all patients were referred to us by a constant

315 group of gynecologic practices, which are being served by our units. Therefore,

316 ascertainment bias is unlikely as is self-selection of patients. Lastly, according to trial

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317 design, only three investigators performed the study procedures. All of them were

318 experienced colposcopists thus excluding bias due to learning curve effects.

319

320 Conclusion

321 In summary, our study defines the proper timing of the AA test during colposcopy (i.e.

322 to assess acetowhite lesions 1 minute after application of AA). Colposcopy and the

323 AA test are widely used by gynecologists and are clinically important tools in the

324 management of women with cervical dysplasia. While the results of our study are not

325 practice changing, they may, however, be helpful in using the AA test in a more

326 comprehensive and efficient way and should therefore be incorporated into clinical

327 practice as well as into guideline recommendations.

328

329 ACKNOWLEDGEMENTS

330 None.

331

332 REFERENCES

333 1. Bornstein J, Bentley J, Bösze P, et al. 2011 colposcopic terminology of the

334 International Federation for Cervical Pathology and Colposcopy. Obstet Gynecol

335 2012;120(1):166–72.

336 2. Waxman AG, Conageski C, Silver MI, et al. ASCCP Colposcopy Standards: How

337 Do We Perform Colposcopy? Implications for Establishing Standards. J Low Genit

338 Tract Dis 2017;21(4):235–41.

339 3. Kühn W, Gieseking F. Die aktuellen Empfehlung der AG-CPC zur Kolposkopie

340 2015. Deutsche Gesellschaft für Gynäkologie und Geburtshilfe, Arbeitsgemeinschaft

341 Zervixpathologie und Kolposkopie e.V. Available at: https://www.ag-

342 cpc.de/empfehlungen/. Accessed Sep 29, 2019.

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343 4. Hinselmann H. Verbesserung der Inspektionsmöglichkeiten von Vulva, Vagina und

344 Portio. Münchener Med Wochenschr 1925;72:1733.

345 5. Pogue BW, Kaufman HB, Zelenchuk A, et al. Analysis of acetic acid-induced

346 whitening of high-grade squamous intraepithelial lesions. J Biomed Opt

347 2001;6(4):397–403.

348 6. Vahedpoor Z, Behrashi M, Khamehchian T, Abedzadeh-Kalahroudi M, Moravveji

349 A, Mohmadi-Kartalayi M. Comparison of the diagnostic value of the visual inspection

350 with acetic acid (VIA) and Pap smear in cervical cancer screening. Taiwan J Obstet

351 Gynecol 2019;58(3):345–8.

352 7. Massad LS, Jeronimo J, Katki HA, Schiffman M. The accuracy of colposcopic

353 grading for detection of high-grade cervical intraepithelial neoplasia. J Low Genit

354 Tract Dis 2009;13(3):137–44.

355 8. Li W, Venkataraman S, Gustafsson U, Oyama JC, Ferris DG, Lieberman RW.

356 Using acetowhite opacity index for detecting cervical intraepithelial neoplasia. J

357 Biomed Opt 2009;14(1):14020.

358 9. Sankaranarayanan R, Nene BM, Shastri SS, et al. HPV screening for cervical

359 cancer in rural India. N Engl J Med 2009;360(14):1385–94.

360 10. Cervix Cancer Screening. Lyon: IARC Press; 2005. (IARC Handbooks of Cancer

361 Prevention; vol. 10).

362 11. Sankaranarayanan R, Nene BM, Dinshaw KA, et al. A cluster randomized

363 controlled trial of visual, cytology and human papillomavirus screening for cancer of

364 the cervix in rural India. Int J Cancer 2005;116(4):617–23.

365 12. Faul F, Erdfelder E, Lang A-G, Buchner A. G*Power 3: A flexible statistical power

366 analysis program for the social, behavioral, and biomedical sciences. Behav Res

367 Methods 2007;39(2):175–91.

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368 13. Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research

369 electronic data capture (REDCap) - a metadata-driven methodology and workflow

370 process for providing translational research informatics support. J Biomed Inform

371 2009;42(2):377–81.

372

373

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374 TABLES

375 Table 1. Patient characteristics

Patient characteristic Value

Number of patients 300
Age (years) 35.2 (29.4 – 44.3); range 19.8 – 78.4
Body mass index (kg/m2) 23.7 (21.3 – 27.3) [44]
Parity 1 (0 – 2) [44]
Allergy (yes / no) 94 (37.2%) / 159 [47]
Smoking (yes / no) 98 (37.8%) / 161 [41]
Alcohol abuse (yes / no) 7 (2.4%) / 288 [5]
Drug abuse (yes / no) 10 (3.5%) / 274 [16]
Concomitant disease (yes / no) 103 (41.2%) / 147 [50]
Prescription drug use (yes / no) 118 (48.8%) / 124 [58]
Immunosuppressive conditions (yes / 4 (1.6%) / 247 [49]
no)

376 Values are counts (percentage proportions) or medians (interquartile ranges).

377 Numbers in square brackets indicate the number of missing values.

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378 Table 2. Colposcopy and histopathology

Item N (%)
Type of transformation zone
Type 1 195 (65.0%)
Type 2 58 (19.3%)
Type 3 47 (15.7%)
Indication for colposcopy (HPV, Pap)
Persistent HPV 32 (10.7%)
ASC-US 11 (3.7%)
ASC-H 21 (7.0%)
AGC-NOS 2 (0.7%)
AGC favor neoplasia 10 (3.3%)
LSIL 69 (23.0%)
HSIL 146 (48.6%)
AIS 8 (2.7%)
Squamous cell carcinoma 1 (0.3%)
HPV status (positive / negative / unknown) 118 (39.3%) / 11 (3.7%) / 171 (57.0%)
Colposcopic findings (before/after acetic acid) before 1 minute 3 minutes 5 minutes
[36]
Normal 221 (73.7%) 40 (13.3%) 43 (14.3%) 61 (23.1%)
Minor changes n/a 142 (47.3%) 137 (45.7%) 107 (40.5%)
Major changes n/a 110 (36.7%) 114 (38.0%) 91 (34.5%)
Non-specific 6 (2.0%) 1 (0.3%) 1 (0.3%) 0 (0.0%)
Suspicious for invasion 2 (0.7%) 2 (0.7%) 2 (0.7%) 1 (0.4%)
Miscellaneous findings 29 (9.7%) 1 (0.3%) 1 (0.3%) 3 (1.1%)
Other 42 (14.0%) 3 (1.0%) 2 (0.7%) 1 (0.4%)
Histological results
Negative for dysplasia 78 (26.0%)
LSIL 65 (21.6%)
HSIL 150 (50.0%)
AIS 2 (0.7%)
Invasive cancer 5 (1.7%)

379 Values are counts (percentage proportions). Numbers in square brackets indicate the

380 number of missing values. AIS, adenocarcinoma in situ; AGC, atypical glandular

381 cells; AGC-NOS, AGC not otherwise specified; ASC, atypical squamous cells; ASC-

382 H, ASC - cannot exclude HSIL; ASC-US, ASC of undetermined significance; HPV,

383 human papilloma virus; HSIL/LSIL, high/low grade squamous intraepithelial lesions;

384 n/a, not applicable, Pap, Pap smear.

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385 Table 3. Primary and secondary study outcomes

Outcome N (%)
Most severe diagnosis found (before / after application of acetic acid)
before acetic acid 73 / 300 (24.3%)
after 1 minute 290 / 300 (96.7%)
after 3 minutes 290 / 300 (96.7%)
after 5 minutes [36] 233 / 264 (88.3%)
Increase in severity of diagnosis
before acetic acid to 1 minute 222 / 300 (74.0%)
1 minute to 3 minutes 8 / 300 (2.7%)
3 minutes to 5 minutes [36] 0 / 264 (0.0%)
Decrease in severity of diagnosis
before acetic acid to 1 minute 2 / 300 (0.7%)
1 minute to 3 minutes 8 / 300 (2.7%)
3 minutes to 5 minutes [36] 22 / 264 (8.3%)

386 Values are counts (percentage proportions). Numbers in square brackets indicate the

387 number of missing values.

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388 Table 4. Video evaluation

Group Time after acetic acid application to …

most severe diagnosis maximum staining intensity start of fading

Overall (N=76 videos) 13.5 (3 – 27.25) 50 (29 – 76.75) 191 (120 – 295)
Grouped by rater
Rater 1 14.5 (1.25 – 28.75) 43.5 (28.25 – 70.0) 210 (120 – 300)
Rater 2 16 (5.5 – 30) 67 (47 – 89.5) 179 (127 – 246)
Rater 3 10 (1 – 20) 48 (39 – 62) 213 (115.5 – 300)
Grouped by histopathology
No pathology (N=15) 15 (3 – 22) 50 (40 – 73) 210 (110 – 300)
LSIL (N=24) 19 (9 – 39.5) 60 (44.75 – 81.5) 212.5 (146.5 – 300)
HSIL (N=34) 7 (1 – 20); P<0.001 vs. LSIL 46 (32.25 – 70.75); P=0.039 vs. LSIL 179.5 (110 – 253.25); P=0.044 vs LSIL
Carcinoma (N=3) 29 (14 – 30) 72.5 (46.75 – 87.5) 248 (218.5 – 296)

389 Values are counts (percentage proportions) or medians (interquartile ranges) of times in seconds. LSIL/HSIL, low/high grade

390 squamous intraepithelial lesions. Statistics for histopathology groups: ANOVA on ranks with Dunn’s pairwise comparison (using data

391 from all raters).

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392 FIGURE LEGENDS

393 Figure 1. Flow diagram of the study and analysis.

394

395 Figure 2. Assessment of acetowhite fading in video colposcopy recordings. (A)

396 Waterfall plots showing time points of fading in low-grade (LSIL, left side) and high-

397 grade (HSIL) lesions (right side) as determined by three raters. (B) Plots of the

398 proportions of not faded acetowhite low-grade (LSIL, open circles) and high-grade

399 (HSIL, filled circles) lesions (fading times averaged over the three raters) versus time

400 after acetic acid application at a resolution of 15 seconds. P-value: Mann-Whitney U

401 rank sum test.

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 RECRUITMENT
Excluded: N=5
Patients asked to participate: N=310
• Declined to participate
Patients included: N=305

COLPOSCOPY
• Start video recording
• Classification of tissue before
application of acetic acid
• Application of actetic acid
• Classification after 1 minute
• Classification after 3 minutes
• Classification after 5 minutes
• Stop video recording
Colposcopies performed: N=305

Excluded: N=5
DATA ANALYSES • Duplicate recruitment (N=4)
Included in analysis: N=300 • Study documentation
missing (N=1)

VIDEO EVALUATION
Random selection video recordings
in a 1:2:2 ratio according to histology
(negative, LSIL, HSIL): N=85
Assessment by three colposcopists:
• Time to most severe diagnosis
• Time until maximum staining intensity
• Time until fading

Excluded: N=9
VIDEO ANALYSES • Poor video quality (N=6)
Included in analysis: N=76 • Data from 1 rater only (N=3)
 A B 100
LSIL HSIL

80 P = .016

Proportion not faded (%)

Individual observations

60

40

20

HSIL
LSIL
0
300 200 100 0 100 200 300 0 100 200 300
Time (seconds) Time (seconds)