Accepted Manuscript

Title: Commercially available molecular tests for human

papillomaviruses (HPV): 2015 update

Author: Mario Poljak Boštjan J. Kocjan Anja Oštrbenk Katja

Seme

PII: S1386-6532(15)00728-3
DOI: http://dx.doi.org/doi:10.1016/j.jcv.2015.10.023
Reference: JCV 3467

To appear in: Journal of Clinical Virology

Received date: 12-9-2015

Revised date: 26-10-2015
Accepted date: 29-10-2015

Please cite this article as: Poljak Mario, Kocjan Boštjan J, Oštrbenk Anja, Seme
Katja.Commercially available molecular tests for human papillomaviruses (HPV): 2015
update.Journal of Clinical Virology http://dx.doi.org/10.1016/j.jcv.2015.10.023

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 Commercially available molecular tests for human

papillomaviruses (HPV): 2015 update

Mario Poljak*, Boštjan J. Kocjan, Anja Oštrbenk, Katja Seme

Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana,

Ljubljana, Slovenia

*Corresponding author: Tel.: +386 1 543 7453; Fax: +386 1 543 7401; E-mail: mario.poljak@mf.uni-

lj.si (M. Poljak)

Highlights

 At least 193 commercial HPV tests and 127 variants of the original tests are available.
 There was a 54% increase in the number of HPV tests in comparison with 2012.
 All but two commercial HPV tests target alpha-HPV types only.
 Only 35% of tests have performance evaluations published in peer-reviewed literature.
 Manufacturers should invest greater effort into evaluating their products.

Abstract

Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in

cervical carcinoma screening and management of women with cervical precancerous lesions as well

as important research tools for epidemiological studies, vaccine development, and implementation

and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified

193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015,

which represents a 54% and 79% increase in the number of distinct HPV tests and variants,

respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were

provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV

tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at

least one published study in peer-reviewed literature has increased significantly in the last three years,

several published performance evaluations are still not in line with agreed-upon standards in the HPV

community. Manufacturers should invest greater effort into evaluating their products and publishing

1
validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented

external quality-control panels and initiatives are required. For evaluating the analytical performance

of the entire range of HPV tests currently on the market, more diverse and reliable external quality-

control programs based on international standards for all important HPV types are indispensable. The

performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative

clinical specimens. In addition, more complete HPV assays containing validated sample-extraction

protocols and appropriate internal controls are urgently needed. Provision of a broader range of

automated systems allowing large-scale HPV testing as well as the development of reliable, rapid,

and affordable molecular point-of-care tests are priorities for the further improvement of HPV tests.

Keywords: human papillomaviruses, HPV, test, screening, cervical cancer

2
 1 1. Background

3 Human papillomaviruses (HPVs) are small, non-enveloped DNA viruses etiologically implicated in

4 the development of several benign and malignant neoplasms of the mucosa and skin [1]. HPVs are

5 hierarchically classified into types, species, and genera [2,3]. As of October 26, 2015, 201 different

6 HPV types, ranging from HPV1 to HPV205, were officially recognized: 65 Alphapapillomaviruses

7 (Alpha-PVs), 51 Betapapillomaviruses (Beta-PVs), 81 Gammapapillomaviruses (Gamma-PVs), three

8 Mupapillomaviruses (Mu-PVs), and a single Nupapillomavirus (Nu-PV)

9 (http://www.hpvcenter.se/html/refclones.html). Three previously recognized HPV types (HPV46,

10 HPV55 and HPV64) were recently re-classified as subtypes (respectively of HPV20, HPV44 and

11 HPV34) while HPV79 was replaced by HPV91 [4]. Several additional HPVs have been completely

12 sequenced, mainly using next-generation sequencing, but are not yet officially recognized and are

13 listed in the PapillomaVirus Episteme (PaVE) database, together with the sequences of animal PV

14 genomes [5]. Alpha-HPV genus is considered the clinically most important HPV genus. Twelve to 25

15 alpha-HPV types are etiologically associated with various human cancers [6,7]: these so-called high-

16 risk or carcinogenic HPV types play the leading etiological role in the development of cervical, anal,

17 and vaginal cancers and their immediate precursors and in a substantial proportion of penile, vulvar,

18 and oropharyngeal (tonsillar) cancers [1,3,8].

19 HPV infection is usually identified by detection of viral nucleic acids in clinical specimens. Soon

20 after identification of the first HPV types, researchers started developing molecular tests for their

21 detection. Thus, several in-house HPV tests and protocols have been successfully used in research

22 laboratories worldwide for more than 25 years, and some of them have been used in key randomized-

23 controlled clinical trials that have proved the clinical usefulness of HPV testing and in randomized-

24 controlled clinical trials of prophylactic HPV vaccines [reviewed in 9-15]. The first commercial HPV

25 tests were developed in the early 1980s, soon after solid evidence was provided that HPV testing

26 could become important in cervical carcinoma screening and management of women with cervical

27 precancerous lesions [reviewed in 12,16], followed by very rapid development of a range of

3
28 commercial HPV tests in the last decade. However, in-house HPV tests are still indispensable in many

29 settings. Because currently available commercial HPV tests almost exclusively target only a limited

30 fraction of the clinically most important alpha-HPV types [17,18], in-house HPV tests are essential for

31 detecting clinically less important alpha-HPV types, HPV types belonging to non-alpha HPV genera,

32 and recently identified HPV types (HPV92 and beyond) and for identifying novel HPV types [19,20].

33 In addition, many laboratories also successfully use in-house HPV tests for diagnostic purposes,

34 mostly in countries/regions that allow such practices, although the great majority of diagnostic

35 laboratories offering routine HPV testing today rely exclusively on commercial HPV tests.

36 In 2010 we published the first comprehensive inventory of commercial molecular tests for detecting

37 alpha-HPVs [17]. At that time we identified 70 commercial HPV assays on the market, and 33 of them

38 were described in detail in our review, including a critical summary of major studies describing their

39 analytical and clinical performance [17]. In the second review, published in 2012, we provided a

40 detailed update for the 16 most clinically relevant HPV assays at that time, identified the most

41 important drawbacks and future challenges of commercial HPV assays, and performed an additional

42 in-depth search to identify as many HPV assays available on the global market as possible [18]. Thus,

43 in 2012 we identified at least 125 commercial HPV tests and 71 variants of the original tests, but

44 unfortunately only a small subset of tests had documented clinical performance and three-quarters of

45 the tests on the market were without a single peer-reviewed publication [18].

46 Here we provide a 2015 update inventory of commercial molecular HPV tests currently present on

47 the market. This review, like the two preceding reviews [17,18], is restricted to tests that identify

48 infection with HPV through detection of their nucleic acids (molecular tests). Different HPV-based or

49 non–HPV-based tests for biomarkers of cervical cancer and/or their immediate precursors that can

50 be used as screening and triage tests are not addressed in this inventory; instead, excellent recent

51 reviews on the topic are available [21-23].

52

53 2. Identification and classification of commercial molecular HPV tests

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54 For this review, data were retrieved from our internal files and from a detailed search using

55 Medline/Pubmed, Web of Science, Scopus, Bing, Google Scholar, and Google, without language or

56 period restrictions. The initial web search was performed in June 2015 and repeated again in late

57 August 2015. In addition, abstracts of most important HPV-related conferences held from 2010 to

58 2015 were carefully reviewed and all possible vendors were repeatedly contacted. Although we did

59 our best, due to the rapid development in commercial HPV tests we likely did not identify all HPV tests

60 currently available on the global market and the omission of any particular available HPV test was

61 unintentional. As summarized in Table 1 (full list of distinct HPV tests) and Table S1 (extended list

62 including all test variants), we identified at least 193 distinct commercial HPV tests and, in addition,

63 at least 127 variants of the original tests currently present on the market. For the purpose of this

64 review, a particular HPV test was considered a variant if it was technologically identical or very similar

65 to the original test but targeted different HPV type(s). For example, the test HPV TS 16 PCR-DEIA

66 (Labo Bio-medical Products, Ev Rijswijk, Netherlands) was considered a distinct test (Table 1) and

67 three other similar tests listed in Table S1 targeting different HPV types (HPV TS 18 PCR-DEIA, HPV

68 TS 31 PCR-DEIA, and HPV TS 45 PCR-DEIA; Labo Bio-medical Products, Ev Rijswijk, Netherlands)

69 were considered variants.

70 Among the 193 distinct commercial HPV tests available on the market in 2015, all but two (Tables

71 1-6 and S1-S6) target alpha-HPV types only. The categorization of alpha-HPV types into different risk

72 categories is extremely challenging, especially for weakly carcinogenic and rare HPV types [6,24,25].

73 In this review, as in the two preceding reviews [17,18], 12 HPV types (HPV16, HPV18, HPV31,

74 HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59) are considered to be

75 carcinogenic (class I) or high-risk HPV (hr-HPV) types, as suggested by the International Agency for

76 Research on Cancer (IARC) [7,24]. These 12 HPV types are referred to as IARC-2009 hr-HPV types

77 in this manuscript. HPV68 is considered probably carcinogenic and 12 additional HPV types possibly

78 carcinogenic: HPV26, HPV30, HPV34, HPV53, HPV66, HPV67, HPV69, HPV70, HPV73, HPV82,

79 HPV85, and HPV97.

80 As summarized in Tables 1-6 and S1, the available commercial HPV tests were provisionally

81 divided for the purpose of this review into eight main groups and several subgroups. Due to space

5
 82 constraints and the availability of excellent recent reviews on the topic, only the general characteristics

83 of the main groups and subgroups of HPV tests were briefly described, but no particular HPV tests.

84 For in-depth descriptions of the most widely used HPV tests, including a critical summary of the major

85 studies describing their analytical and clinical performance, we refer to our previous reviews published

86 in 2010 and 2012 [17,18] and other recently published reviews [9-16,21,22,26-37]. For each

87 group/subgroup of HPV tests, a brief aggregative summary of the available performance data was

88 provided: for tests designed to be used for major agreed-upon clinical indications for HPV testing, we

89 provided US Food and Drug Administration (FDA) approval status and compliance with validation

90 criteria for HPV-based primary screening based on the recent meta-analysis [32] and for HPV

91 genotyping tests, we provided their performance on several rounds of World Health Organization

92 (WHO) HPV LabNet genotyping proficiency panels containing defined amounts of the international

93 standards and candidate standards for the clinically most important HPV types [38,39].

94

95 3. General characteristics of the main groups and subgroups of available commercial

96 HPV molecular tests

97

98 3.1. hr-HPV DNA screening tests

99

100 Hr-HPV DNA screening tests comprise a group of qualitative or semi-quantitative assays that utilize

101 various technologies to identify the presence or absence of targeted carcinogenic HPV types. The

102 results are reported as positive or negative for the targeted HPV types without determination of the

103 exact HPV type. For the purpose of this review, based on the targeted HPV types, hr-HPV DNA

104 screening tests were divided into four subgroups (Tables 1 and S1). The most widely used HPV tests

105 with abundant clinical performance data belong to the subgroup of tests that target IARC-2009 hr-

106 HPV types plus HPV66 and/or HPV68 [32]. Seven distinct HPV tests were identified in this group. The

107 three tests from the second subgroup target 12 IARC-2009 hr-HPV types only, and the 13 tests from

108 the third subgroup target 12 IARC-2009 hr-HPV types and some additional alpha-HPV types. Six tests

6
109 from the last subgroup screen for the great majority of, but not all, IARC-2009 hr-HPV types. Out of

110 29 hr-HPV DNA screening tests in this group, two are currently approved by the US FDA (Hybrid

111 Capture 2 (HC2) HPV DNA Test; Cervista HPV HR Test) and at least two additional tests (EIA kit

112 HPV GP HR; HPV-Risk assay) are considered clinically validated for use in primary cervical cancer

113 screening [32].

114

115 3.2. hr-HPV DNA screening tests with concurrent or reflex partial genotyping for the main hr-HPV

116 types

117

118 In recent years, partial HPV genotyping has been evaluated for its clinical potential to improve

119 screening accuracy and efficiency, and has been shown to identify women at highest risk for cervical

120 disease in numerous studies [8,33,40]. As a result, novel HPV tests with concurrent or reflex partial

121 genotyping capabilities to detect IARC-2009 hr-HPV and distinguish types with exceptionally high

122 oncogenic potential (mainly HPV16/18) have been designed and approved for use in various

123 countries. These tests are perfectly suited for countries that use or will use partial genotyping as a

124 main or complementary reflex method for HPV screen-positive women. According to the most recent

125 European guidelines for cervical cancer screening [41] and protocols announced in European

126 countries moving toward the HPV primary screening, cytology will be used as the main reflex method

127 for hr-HPV screen-positive women in Europe, in contrast to the United States, where recent interim

128 clinical guidelines for cervical cancer screening favor triage of hr-HPV screen-positive women using

129 a combination of HPV16/18 typing and reflex cytology for women positive for the 12 other hr-HPV

130 types [42]. Similarly, Australian HPV-based primary screening, which will start in May 2017, will triage

131 with partial HPV genotyping and reflex cytology [43]. Among 24 tests from this group, in 17 tests

132 partial genotyping for the main hr-HPV is performed concurrently and in seven tests as reflex

133 genotyping (Tables 2 and S2). From this group of tests, two (cobas 4800 HPV Test; Cervista HPV

134 16/18 Test) are currently approved by the US FDA and at least two additional tests (RealTime High

135 Risk HPV test; BD Onclarity HPV Assay) are considered clinically validated for use in primary cervical

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136 cancer screening [32]. The cobas 4800 HPV test is the first HPV test approved by the US FDA as

137 stand-alone cervical cancer screening test [33].

138

139 3.3. HPV DNA full genotyping tests

140

141 HPV DNA full genotyping tests still represent the largest group of commercially available HPV

142 tests. The great majority of these tests have no role in screening or clinical management at present,

143 but standardized genotyping tests are well-established research tools and are invaluable for vaccine

144 development, implementation, and monitoring of vaccination programs, for epidemiological studies,

145 and for testing clinical samples with degraded nucleic acids [22,28]. In contrast to the first two groups

146 of HPV tests, no consensus exists on how to assess the performance characteristics of HPV

147 genotyping tests at present. However, Schutzbank and Ginocchio recently proposed a procedure for

148 assessing the clinical and analytical performance characteristics of an HPV genotyping test [44], WHO

149 HPV LabNet genotyping proficiency panels were developed and successfully used in five rounds of

150 global proficiency studies of HPV genotyping tests [38,39], and the VALGENT initiative was launched

151 for comprehensive validation and comparison of HPV genotyping tests [31].

152 For the purpose of this study, a particular HPV test was classified as a full genotyping test only if

153 all IARC-2009 hr-HPV types could be distinguished in a single reaction. As summarized in Tables 3

154 and S3, we identified 68 distinct full genotyping HPV tests and 17 variants and we divided them into

155 nine subgroups based on the technology used for genotyping the targeted HPV types.

156 Tests that utilize the principle of reverse hybridization are the largest and most commonly used

157 subgroup of HPV DNA full genotyping tests. In all versions, PCR amplification of a fragment of the

158 HPV genome is followed by denaturation of the resulting amplicons and detection with HPV type-

159 specific probes immobilized on a strip, filter, or microtiter-wells. Among 20 distinct HPV tests and

160 seven variants from this subgroup, analytical performance of six, nine, and nine distinct tests were

161 evaluated using WHO HPV LabNet proficiency panels in 2010, 2011, and 2013, respectively

162 [38,45,46].

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163 Real-time PCR based full genotyping tests are type-specific assays designed to amplify and

164 simultaneously detect or quantify the targeted DNA. Tests belonging to this subgroup use non-specific

165 fluorescent dyes that intercalate with any double-stranded DNA or sequence-specific DNA probes for

166 detecting specific amplicons. Among 10 distinct HPV tests and four variants from this subgroup,

167 analytical performance for one, one, and four distinct tests were evaluated using WHO HPV LabNet

168 proficiency panels in 2010, 2011, and 2013, respectively [38,45,46].

169 Medium- or low-density microarray tests are based on a similar principle to that of reverse line-blot

170 assays; however, detecting the immobilized PCR products employs the principle of fluorescence

171 detection or chromogenic precipitation. Among 16 distinct HPV tests and four variants from this

172 subgroup, analytical performance for five, six, and five distinct tests were evaluated using WHO HPV

173 LabNet proficiency panels in 2010, 2011, and 2013, respectively, [38,45,46] and in addition one test

174 (PapilloCheck High-risk Test) is considered clinically validated for use in primary cervical cancer

175 screening [32].

176 Microsphere bead–based tests are a highly specific and sensitive subgroup of full genotyping tests

177 [47]. This technology is based on PCR amplification, followed by the detection of amplicons with HPV

178 type-specific polystyrene or digitally barcoded photolithographic beads. Among nine distinct HPV tests

179 and single variant from this subgroup, analytical performance for three, one, and one distinct tests

180 were evaluated using WHO HPV LabNet proficiency panels in 2010, 2011, and 2013, respectively

181 [38,45,46].

182 The remaining five subgroups comprise a relatively small number of HPV DNA full genotyping

183 tests. They are all based on classical PCR amplification but use various methods for genotyping IARC-

184 2009 hr-HPV types and several other HPV types. In this review, we identified four distinct tests that

185 genotype PCR products using gel electrophoresis, three tests using capillary electrophoresis, two

186 tests using matrix-assisted laser desorbtion/ionization time-of-flight mass spectrometry, three distinct

187 tests and a single variant using sequencing, and a single test using loop-mediated isothermal

188 amplification and an electrochemical DNA chip for detecting specific amplicons. Only a single test

189 from these five subgroups had been evaluated using WHO HPV LabNet proficiency panels in three

190 survey rounds.

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191

192 3.4. HPV DNA type- or group-specific genotyping tests

193

194 HPV DNA type- or group-specific tests are a group of tests capable of detecting only a subset of

195 IARC-2009 hr-HPV types and on several occasions only a single or two IARC-2009 hr-HPV types. It

196 is currently the second-largest group of HPV tests with numerous variants that we divided into four

197 subgroups based on the technology used for genotyping the targeted HPV types (Tables 4 and S4).

198 The largest subgroup, based on real-time PCR technology, comprises 22 distinct HPV tests and 33

199 variants. However, only three tests from this subgroup were evaluated using the 2013 WHO HPV

200 LabNet proficiency panel. Similarly, only a single test from the other three subgroups was evaluated

201 using one WHO HPV LabNet proficiency panel [38,45,46].

202

203 3.5. hr-HPV E6/E7 mRNA tests

204

205 Although most of the currently available HPV tests are DNA-based, several studies have shown

206 that testing for HPV mRNA can be clinically useful due to its higher specificity [34,36,48,49]. For

207 diagnostic purposes, the mRNA transcripts of the most significant viral oncoproteins E6 and E7 are

208 detected by reverse-transcriptase PCR or nucleic acid sequence-based amplification (NASBA). In this

209 review, nine distinct HPV tests and a single variant were identified (Tables 5 and S5). From this group,

210 one HPV test (APTIMA HPV assay) is currently approved by the US FDA and can also be considered

211 clinically validated for use in primary screening once longitudinal data is available [32].

212

213 3.6. in situ hybridization DNA-based HPV tests

214

215 In situ hybridization is the only molecular method that enables detection and visualization of HPV

216 DNA in a morphological content. In this inventory, 27 distinct HPV tests and 29 variants were identified

217 (Tables 6 and S6).

218

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219 3.7. in situ hybridization mRNA-based HPV tests

220

221 Two HPV tests classified in this group utilize technology that combines detection of viral mRNA

222 and in situ hybridization (Tables 6 and S6). Following hybridization with E6, E7 mRNA-specific

223 oligonucleotide, the detection and quantification of the HPV E6 and E7 mRNA transcripts is performed

224 using flow cytometry.

225

226 3.8. HPV DNA tests targeting miscellaneous HPV types

227

228 Both tests from this group (RHA Kit Skin (beta) HPV; Diassay, Rijswijk, The Netherlands and LMNX

229 kit HSL-PCR; Labo Bio-medical Products, Ev Rijswijk, The Netherlands) utilize the same principle of

230 reverse hybridization, where PCR amplification is followed by amplicon denaturation and hybridization

231 with HPV type-specific probes immobilized on a strip. The first test allows detection of a subset of skin

232 cancer–associated beta-HPV types, and the second test allows detection of 22 HPV types from alpha-

233 , gamma-, mu-, and nu-PV genera commonly associated with the development of skin warts.

234

235 4. Discussion

236

237 Our 2015 inventory of commercial HPV tests showed the existence of at least 193 distinct HPV

238 tests and at least 127 test variants currently present on the market, which represents a 54.4% and

239 78.8% increase in distinct HPV tests and variant numbers, respectively, compared to 2012. Although

240 we predicted a substantial increase in the number of HPV tests in both previous inventories [17,18],

241 we were surprised by such rapid growth in the last three years. However, when estimating the number

242 of different HPV kits currently on the global market, one should bear in mind all the limitations of our

243 inventory. Although we have tried to do our best, it is very likely that our 2015 estimation is very

244 conservative. The greatest underestimation is probably in Southeast Asia, mainly due to the

245 availability of manufacturers’ webpages in local languages only, lack of transparent webpages, and

11
246 indirect marketing of tests (by second-level traders) and consequent uncertainty about the actual

247 manufacturer of the certain test. In this rapidly changing market, new companies and mergers also

248 frequently cause additional uncertainty. Other issues in building and updating an HPV test database

249 are the almost exclusive orientation of the majority of HPV test researchers towards the Western

250 market, their unawareness of the situation on emerging markets, and the existence of a publication

251 bias (at both the editorial and reviewer levels) toward products developed and manufactured in the

252 non-Western world. Therefore one can hardly judge how accurate the insight is that this inventory

253 provides into the current global market of HPV tests. As an example, while compiling the database

254 we found that HPV tests produced by the Hybribio company (Beijing, China) are officially approved

255 for use in the cervical cancer prevention program in China, that the company had 2012 annual sales

256 of 1.5 million HPV tests in mainland China only, and that the company provided 300,000 free-of-

257 charge tests to the Chaozhou provincial government in 2009–2011. All of these findings are mainly

258 unknown to the HPV community and the great majority of HPV researchers are not even aware of the

259 existence of Hybribio company.

260 Despite all of these limitations and obstacles, we have provided here the first overview of

261 manufacturers’ distribution by continent and country according to the number of different HPV tests

262 currently on the market. Considering all biases and calculating only the main HPV tests without their

263 variants, to our surprise the largest number of HPV tests is currently produced in Europe (47%),

264 followed by Asia (31%) and North America (21%). Surprisingly, we found only one HPV test

265 manufacturer in Australia and none in Latin America and Africa. By country criteria, the largest number

266 of HPV tests is currently produced in United States (18.3%), followed by South Korea (16.7%), the

267 Netherlands (11.5%), China (10.5%), Germany (10.0%), Italy (7.8%), and Russia (7.8%). It has to be

268 pointed out that, if considering the number of tests sold instead of the number of different tests

269 manufactured in a given country/continent, the figures would probably be different.

270 Another surprising finding of our three inventories is extensive intra-manufacturer dynamics.

271 Several companies are constantly changing the design and names of their tests, resulting in delayed

272 and non-updated data presented on vendors’ webpages. The 2015 update of the inventory of HPV

273 tests available on the market was thus not a simple process in which we simply added newly

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274 developed tests to the 2012 list of HPV tests [18]. Instead, the existence of all HPV tests listed in

275 Tables 1 and S1 was again double-checked with manufacturers. Aiming to provide as accurate names

276 of current products present on the market as possible, we learned that one cannot simply rely on data

277 presented on the manufacturers’ official homepages or in scientific publications, but must complement

278 this information with repeated contacts with responsible people in several diagnostic companies,

279 mainly those not regularly present at major HPV-related national and international conferences. To

280 our experience, finding the “right” and longstanding person in a particular company to address

281 questions to and obtain reliable data from is the main challenge in building such a database. Having

282 regularly monitored changes in the HPV test market during the last 6 years, we built a close

283 relationship with key individuals in several non-Western world companies producing and successfully

284 selling a range of HPV tests around the globe. This is the main reason why we strongly believe that

285 we built the most accurate inventory of HPV tests on the global market possible.

286 With such an exceptionally high number of commercial HPV tests on the market, alpha-HPVs are

287 currently still among most attractive microbial targets for molecular diagnostic companies.

288 Unfortunately, today’s HPV test global market is one of the most confusing and least regulated, and

289 with most divergent diagnostic products on the market, sometimes colloquially described as the “Wild

290 West.” In addition, we can predict with high certainty that the number and diversity of commercial HPV

291 tests will continue to increase over the next 5 years due to the promising marketing opportunities for

292 manufacturers around the world. Namely, most “traditional” molecular diagnostic microbiology testing

293 areas with proven clinical utility (e.g., human immunodeficiency virus, hepatitis B and C viruses) are

294 very mature, with expected annual growth rates settling around 3 to 5% [50]. Recent analysis has

295 shown the outlying position of HPV tests on the global market, with the expected compound annual

296 growth rate (CAGR) as high as 20% [50], or in another forecast over 70% from 2013 to 2019

297 (http://www.transparencymarketresearch.com). The total global cervical cancer diagnostic test market

298 was valued at USD 5.9 billion in 2013 and is expected to grow at a CAGR of 6.1% from 2014 to 2020,

299 to reach an estimated value of USD 8.9 billion in 2020. The global diagnostic testing market for

300 sexually transmitted diseases was valued at USD 65.9 billion in 2012; with expected growth at a high

301 CAGR of 8.1% in the forecast period from 2013 to 2019, the market is anticipated to reach an

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302 estimated value of USD 108.5 billion by the end of 2019

303 (http://www.transparencymarketresearch.com).

304 In our previous review in 2012, we were able to identify a single publication in peer-reviewed

305 literature for less than 25% of HPV tests [18]. This has improved dramatically in the last 3 years

306 because 110/193 (57.0%) of HPV tests on the market in 2015 had at least one publication in peer-

307 reviewed literature as of August 31, 2015. However, only 69/193 (35.7%) of HPV tests on the market

308 in 2015 had a documented performance evaluation (analytical and/or clinical) in peer-reviewed

309 literature as of August 31, 2015 in contrast to the remaining 41 HPV tests, for which only descriptive

310 studies can be found in the literature. The great majority of descriptive studies were cross-sectional,

311 in which a particular HPV test was used for determining HPV prevalence in different target populations

312 (including several non-genital) without providing any data on key test performance characteristics

313 (sensitivity, specificity, reproducibility, etc.). In additionally, even if performance evaluations were

314 published, these were mostly “test A versus test B” approach comparisons without knowing which test

315 is a reference standard, and they were carried out on ad hoc collections of mostly very heterogeneous

316 clinical samples and from patients without any proper follow-up. Often a company published an

317 evaluation of a test that then, because of the lengthy publication process, never appeared on the

318 market. Namely, in the meantime an updated test was already developed and adapted to new market

319 conditions, competition, or constantly changing local and/or international guidelines. Furthermore,

320 inaccurate names of the HPV tests were often used in peer-reviewed publications, resulting in

321 additional confusion. We therefore strongly advise all authors consistently to use accurate names of

322 HPV tests in their publications.

323 Based on the results of this inventory, we urge manufacturers of HPV tests to put substantially

324 more effort into evaluating their current and future products. Although several HPV tests on the market

325 have been marked with the European Conformity mark for In Vitro Diagnostics (CE-IVD) compliant

326 for diagnostic use in Europe, it should be stressed that the CE-IVD marking process is mainly technical

327 and is substantially less demanding in comparison with the US FDA or the Australian Therapeutic

328 Goods Administration evaluation procedure. Although CE-IVD marking is necessary for an HPV test

329 to be legally placed on the European market, in the authors’ experience, CE-IVD marking per se does

14
330 not particularly impress the great majority of laboratory directors or issuers of public tenders when

331 deciding/requesting which HPV test to use. On many occasions, the decision process for which test

332 to use for HPV detection is more complex and should always start by asking what purpose the HPV

333 test is needed for. Thus the two most important parameters that a manufacturer has to define when

334 designing a new HPV test are (i) a set of targeted HPV types and (ii) a level of analytical sensitivity.

335 These parameters define the purpose of the HPV test. Currently three completely different groups of

336 HPV tests are needed for different purposes: (i) major agreed-upon indications for HPV testing in

337 current clinical practice, (ii) for epidemiological and vaccine-related studies, and (iii) for various

338 research purposes. Depending on the intended use of the new test, the manufacturer must also select

339 an appropriate evaluation/validation protocol.

340 If the test is intended to be used for major agreed-upon indications for HPV testing in current clinical

341 practice—that is, (i) management and triage of women with borderline cytological findings; (ii) post-

342 treatment follow-up/test of a cure; (iii) primary cervical cancer screening; and (iv) resolution of

343 diagnostic uncertainties [reviewed in 14,22,51]—we suggest that manufacturers seek advice from

344 established HPV researchers in the very early phase of development on how to properly design a

345 novel test and how to evaluate its clinical performance in such a way that the HPV community will

346 accept its evaluation/validation results. Although we have an increasing understanding of which HPV

347 tests are valid for HPV-based primary cervical cancer screening, given an internationally accepted

348 and applied validation framework and published professional guidance [27,52-54], we do not have

349 widely accepted equivalent metrics to judge the validity of HPV tests in other clinical settings, including

350 post-treatment surveillance and the triage of low-grade abnormalities [27,55]. International efforts to

351 create such validation guidelines will be of great benefit particularly because existing data show

352 significant variation between commercially available tests in clinical settings other than primary HPV-

353 based primary cervical cancer screening [27,55]. One such validation “rule of thumb” for the triage of

354 low-grade abnormalities has been recently proposed by Arbyn et al. [14]. However, to appropriately

355 evaluate and control the performance quality of HPV tests for major agreed-upon clinical indications,

356 we also need carefully designed external quality control panels in addition to the appropriate

357 guidelines. Because the proper function of all testing steps (e.g., nucleic acid extraction, amplification

15
358 and analysis of amplified products) determines the final clinical sensitivity and specificity of HPV

359 testing, when designing external quality panels for evaluating the clinical performance of HPV tests

360 one should produce panel members that mimic clinical samples as much as possible in terms of

361 composition (human cells), transportation medium (with/without fixative), and total volume (greater

362 than the minimum volume requested for preanalytical processing) [56].

363 In contrast, if an HPV test is needed for HPV vaccine development, implementation, and monitoring

364 of vaccination programs, for epidemiological studies, for testing clinical samples with degraded nucleic

365 acids (e.g., formalin-fixed, paraffin-embedded tissues), and for various research purposes, this may

366 require the use of tests with completely different analytical performance characteristics as tests with

367 clinically validated cutoffs. These tests require high analytical sensitivity, high analytical specificity,

368 and a very accurate type-specific resolution of several high- and low-risk HPV types without cross-

369 reactivity [22,57]. Their performance should therefore be evaluated differently than HPV tests for

370 major agreed-upon clinical indications for HPV testing. Currently the best approach is to use WHO

371 HPV LabNet proficiency panels containing defined amounts of the international standards and

372 candidate standards for the clinically most important HPV types, allowing extensive evaluation of

373 analytical performance of a broad range of HPV tests [38,39]. There is also an increasing variety of

374 international and national external quality control programs, which vary in their size, frequency,

375 general scope (clinical vs. analytical performance), and technical scope (broad vs. type-specific

376 performance) [56,58,59]. Major external quality-control programs for HPV tests, the main issues, and

377 evolving requirements for robust quality assurance are described in detail in a separate paper in this

378 supplement [56].

379 As previously mentioned, when evaluating their products many manufacturers have adopted the

380 “test A versus test B” approach. When two tests are compared without knowing which of them is the

381 standard, or when a certain test is used as a standard without actually being a standard, this is not

382 the best evaluation approach. If the “test A versus test B” approach is chosen and the new HPV test

383 is intended for use in current clinical practice, an FDA-approved test or other tests considered clinically

384 validated [32] should be used as a standard. If a new HPV test is intended to be used for non-clinical

385 purposes, then one of the tests that scored the highest in the WHO HPV LabNet proficiency panels

16
386 should be used as a standard. Multi-test comparison studies and initiatives, which provide a more

387 accurate means of comparison of HPV tests, have been launched recently, such as the

388 PREDICTORS studies [60,61] or VALGENT initiative, described in detail in a separate paper in this

389 supplement [31]. We urge manufacturers, until a more diverse range of evaluation and/or external

390 quality control panels for assessing the performance of HPV tests are available, to join such multi-test

391 comparisons studies and initiatives.

392 Despite of the substantial number of commercial HPV tests available, it should be noted that, in

393 contrast to commercial diagnostic assays for “classic” molecular microbiology targets, the vast

394 majority of HPV tests currently on the market are not complete diagnostic assays because they do

395 not contain a sample extraction part and a number of them do not even mention nucleic acid extraction

396 methodology in their manufacturer’s instructions. Because the extraction of nucleic acids is an

397 invaluable part of the entire HPV testing procedure, we urge the manufacturers of HPV tests to place

398 substantially more effort into this initial and crucial step of molecular testing. As for “classic” molecular

399 microbiology targets, the sample extraction procedure should be thoroughly validated by

400 manufacturers for each of the recommended clinical sample types separately and list of validated

401 specimen types, as well as sample collection devices, should be provided in the manufacturer’s

402 instructions. In addition, in contrast to diagnostic assays for “classic” molecular microbiology targets

403 in which control of specimen adequacy and the impact of potential interfering substances using

404 appropriate internal controls has been standard for two decades, only a minority of HPV commercial

405 test on the market have internal controls included to ensure that a negative HPV result is not due to

406 a poorly collected sample or inhibitory substances. Although assay internal controls may not always

407 reflect adequate sampling and do not completely obviate the risk of false negatives, we strongly

408 believe that an HPV test intended to be used for routine HPV detection should have an internal control

409 in place to control for cellularity, particularly if the HPV test is used for self-collected specimens, where

410 the adequacy of sampling is reliant on the participants following the protocols given to them [62].

411 The great majority of complete HPV diagnostic assays currently on the market are validated to be

412 used for testing cervical scrapings only, and on top of that for scrapings collected by a single or usually

17
413 up to no more than three different specimen collection devices. Although few commercial HPV assays

414 have been evaluated, and some of them are specifically designed to test alternative clinical specimens

415 such as self-collected cervicovaginal lavage, vaginal and urethral swabs, urine and tissue specimens

416 (in situ hybridization-based HPV tests), we urgently need an evaluation of the performance of as many

417 commercial HPV tests as possible on a broader range of alternative clinical specimens; for example,

418 other types of self-collected anogenital samples (different brushes, swabs and sponges, tampons),

419 oral swabs, saliva, anal swabs, penile swabs, Guthrie-type filter paper, and so on, and validation of

420 different non–in situ hybridization-based HPV tests with higher analytical sensitivity and specificity

421 and broader HPV type coverage on fresh, frozen, and formalin-fixed, paraffin-embedded tissue

422 specimens [57,63,64]. Thus, instead of developing just another HPV test for the mature clinical market

423 of HPV tests for cervical scrapings we suggest that newcomers or existing vendors with poor market

424 penetration instead focus on evaluation, validation, and/or development of HPV kits for alternative

425 clinical specimens. In our opinion, there will be bright opportunities in the near future in this field. For

426 example, on August 20th, 2015 it was announced that one of a California-based manufacturer

427 received USD 250,000 in funding from US National Institutes of Health to develop a point-of-care

428 rapid molecular diagnostic assay for HPV detection in oral specimens. Although the first HPV-based

429 screening tests designed for use in resource-poor areas are being developed and show encouraging

430 results [21,65], we also need more HPV tests in this field.

431

432 5. Conclusions

433

434 Our inventory identified 193 distinct commercial HPV tests and at least 127 test variants available

435 at the market in 2015, which were provisionally divided into eight main groups and several subgroups.

436 Among the 193 HPV tests, all but two target alpha-HPV types only. Although the number of HPV tests

437 with at least one published study in peer-reviewed literature increased significantly in the last 3 years,

438 several published performance evaluations were not in line with agreed-upon standards in the HPV

439 community, and many of them were descriptive studies only. Manufacturers should invest greater

440 effort into evaluating their products. To achieve this, more clinically oriented external quality-control

18
441 programs and initiatives are required. In additionally, more divergent and reliable external quality-

442 control panels containing international standards for all important HPV types and allowing extensive

443 evaluation of analytical performance of a whole range of HPV tests on the market are needed. We

444 also believe that performance evaluation of a wider range of HPV tests on a variety of alternative

445 clinical specimens is required as well as development of more complete HPV assays containing

446 validated sample extraction protocols and appropriate internal controls. HPV tests in general should

447 be more competitively priced, especially for resource-poor countries. Uniform HPV test-validation

448 criteria (both analytical and clinical) based on international standards should make it possible to issue

449 more competitive and fair tender notices for purchasing. Finally, provision of broader range of

450 automated systems allowing large-scale HPV testing as well as the development of reliable, rapid,

451 and affordable molecular point-of-care tests are priorities for further improving HPV tests.

452

453

454 Disclosed potential conflict of interest

455 The authors’ institution received several research grants from Abbott Molecular. Mario Poljak received

456 reimbursement of travel expenses for attending meetings and conferences from Abbott Molecular,

457 Qiagen and Roche, and honoraria for speaking and consultancy from Abbott Molecular until mid-

458 2014. Mario Poljak declares no competing interest since September 2014. Other authors declare no

459 competing interest.

460

461

462 Acknowledgements

463 Mario Poljak and Anja Oštrbenk are supported by the seventh framework program of DG Research

464 of the European Commission, through the COHEAHR Network (grant no. 603019). Anja Oštrbenk is

465 financially supported through the Young Researcher Training Program funded by the Slovenian

466 Research Agency (ARRS). We are grateful to Anja Šterbenc and Jana Mlakar for their invaluable

467 support with the literature search and technical help.

468
19
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699 Table 1: hr-HPV DNA screening tests present on the market in August 2015.

Tests targeting IARC-2009 hr-HPV types plus HPV66 and/or HPV68

Hybrid Capture 2 (HC2) HPV DNA Test (Qiagen Gaithersburg, Inc., MD, USA)
EIA kit HPV GP HR (Diassay, Rijswijk, The Netherlands)
Cervista HPV HR Test (Hologic, Madison, WI)
CareHPV Test (Qiagen Gaithersburg, Inc., MD, USA)
Amplicor HPV Test (Roche Molecular Systems Inc., Alameda, CA, USA)
13 High-risk HPV Real-time PCR Kit (Hybribio, Beijing, China)
Biorad Dx HR-HPV Auto Assay (Bio-Rad, Hercules, CA, USA)
Tests targeting IARC-2009 hr-HPV types only
HPV High Risk Screen Real-TM Quant (Sacace, Como, Italy; Nuclear Laser Medicine S.R.L., Milano, Italia)
HPV High Risk Screen Real-TM Quant 2 x (Sacace, Como, Italy; Nuclear Laser Medicine S.R.L., Milano,
Italia)
AmpliSens HPV HCR screen-titre-FRT PCR kit (Federal State Institution of Science, Moscow, Russia;
Ecoli, Bratislava, Slovak Republic) and 1 variant
Tests targeting IARC-2009 hr-HPV types and additional alpha-HPV types
HPV-Risk assay (Self-Screen BV, Amsterdam, The Netherlands)
Seeplex HPV4A ACE Screening (Seegene, Seoul, Korea)
Urine-Based HPV (High and Low Risk) PCR Detection Kit (Norgen, Thorold, Canada)
STD Kit (Autoimmun Diagnostika GmbH, Strassberg, Germany)
AmpliSens HPV HCR screen-Eph PCR kit (Federal State Institution of Science, Moscow, Russia; Ecoli,
Bratislava, Slovak Republic) and 1 variant
HPV-DNA Assay Kit (Tofema, Seoul, Korea)
PapilloScreen (GeneMatrix Co., Seoul, Korea)
HPV Screen PCR Kit (BioCore, Seoul, Korea)
AmpliQuality HPV-SM (AB Analitica, Padova, Italy)
AmpliQuality HPV-HS Bio (AB Analitica, Padova, Italy)
Human Papilloma Virus (HPV Common/double check) (Genekam Biotechnology, Duisburg, Germany)

28
 BIOPAP Kit (Biotools, Nave, Spain)
High-risk human papillomavirus DNA Diagnostic kit (Sansure Biotech Inc., Changsha, Hunan, China)
Tests targeting a subset of IARC-2009 hr-HPV types
HPV High Risk Screen (Sacace, Como, Italy; Nuclear Laser Medicine S.R.L., Milano, Italia)
AmpliSens HPV HCR screen-FEP PCR kit (3x) (Federal State Institution of Science, Moscow, Russia;
Ecoli, Bratislava, Slovak Republic)
HPV Total & High Risk (Clonit, Milano, Italy)
Absolute HPV HR Test (BioSewoom, Seoul, Korea)
HPV Screening (Clonit, Milano, Italy)
Cancer Molecular Marker TEST (GoodGene, Seoul, South Korea)
700

29
701 Table 2: hr-HPV DNA screening tests with concurrent or reflex partial genotyping for the main hr-
702 HPV types present on the market in August 2015

hr-HPV DNA screening tests with concurrent partial genotyping for the main hr-HPV types
cobas 4800 HPV Test (Roche Molecular Systems Inc., Alameda, CA, USA)
RealTime High Risk HPV test (Abbott Molecular, Des Plaines, IL)
BD Onclarity HPV Assay (BD Diagnostics, Sparks, MD)
Xpert HPV (Cepheid, Sunnyvale, CA)
14 High-risk HPV with 16/18 Genotyping Real-time PCR Kit (Hybribio, Beijing, China)
Realquality RQ-HPV HR (AB Analitica, Padova, Italy) and 2 variants
GenoFlow HPV-HR screening kit (FT-PRO) (DiagCor Bioscience, Honk Kong, China)
HPV Easy-Screening Kit (Autoimmun Diagnostika GmbH, Strassberg, Germany)
AdvanSure HPV Screening real-time PCR (LGLS Diagnostics, Seoul, Korea)
GenoID Real-Time HPV Assay (GenoID, Budapest, Hungary)
AmpliSens HPV HCR screen-FEP PCR kit (Federal State Institution of Science, Moscow, Russia; Ecoli,
Bratislava, Slovak Republic)
RealLine HPV High Risk, Genotype (Str-format) (Bioron Diagnostics GmbH, Ludwigshafen, Germany)
RealLine HPV High Risk, Genotype quantitative (Str-format) (Bioron Diagnostics GmbH,
Ludwigshafen, Germany)
GenoFlow HPV-HR screening kit (R2-M) (DiagCor Bioscience, Honk Kong, China)
High-Risk Human Papillomavirus (HPV) and Genotyping 16 & 18 Real Time PCR Kit (Liferiver,
Shanghai, China)
HPV 5+9 Test Kit (Tellgen Corporation, Shanghai, China) and 1 variant
RealLine HPV High Risk, Genotype (Fla-format) (Bioron Diagnostics GmbH, Ludwigshafen, Germany)
hr-HPV DNA screening tests with reflex partial genotyping for the main hr-HPV types
Cervista HPV 16/18 Test (Hologic, Madison, WI)
digene HPV Genotyping PS Test, RUO (Qiagen Gaithersburg, Inc., MD, USA)
HPV 16/18 Real-TM Quant (Sacace, Como, Italy; Nuclear Laser Medicine S.R.L., Milano, Italia)
Human papillomavirus 16/18 (Sacace, Como, Italy; Nuclear Laser Medicine S.R.L., Milano, Italia) and 3 variants
AmpliSens HPV 16/18-FRT PCR kit (Federal State Institution of Science, Moscow, Russia; Ecoli s.r.o.,
Bratislava, Slovak Republic) and 1 variant
AmpliSens HPV 16/18-EPh PCR kit (Federal State Institution of Science, Moscow, Russia; Ecoli s.r.o.,
Bratislava, Slovak Republic) and 3 variants
AmpliSens HPV 16/18-FEP PCR kit (Federal State Institution of Science, Moscow, Russia; Ecoli s.r.o.,
Bratislava, Slovak Republic) and 1 variant
703
704

30
705 Table 3: HPV DNA full genotyping tests present on the market in August 2015

Strip, filter or microtiter-well hybridization based full genotyping tests

Linear Array HPV Genotyping Test (Roche Molecular Systems Inc., Alameda, CA, USA)
INNO-LiPA HPV Genotyping Extra II (Fujirebio, Gent, Belgium)
RHA Kit SPF10-LiPA25version 1 (Labo Bio-medical Products, Ev Rijswijk, The Netherlands)
DNA ELISA Kit HPV SPF10version 1 (Labo Bio-medical Products, Ev Rijswijk, The Netherlands)
EasyChip HPV Blot Kit (King Car, Taipei, Taiwan)
REBA HPV-ID (Molecules and Diagnostics, Wonju, Korea)
AmpliQuality HPV-type (AB Analitica, Padova, Italy) and 3 variants
GenoFlow HPV Array Test (DiagCor Bioscience, Honk Kong, China)
HPV Direct-Flow Chip (Master Diagnostika, Granada, Spain; Hospitex Diagnostics, Firenza, Italy)
HPV Easy-Typing Kit (Autoimmun Diagnostika GmbH, Strassberg, Germany)
Full Spectrum HPV Amplification and detection system (GenoID, Budapest, Hungary)
High PapillomaStrip (Operon, Zaragoza, Spain) and 1 variant
HPV Genotyping Lateral Flow Array (DiaProbe Inc, Seoul, Korea)
HPV Genotyping Chip (BioCore, Seoul, Korea)
GenoFlow HPV array test kit (R2-M) (DiagCor Bioscience, Honk Kong, China)
AdvanSure HPV GenoBlot Assay (LGLS Diagnostics, Seoul, Korea)
Genotyping Kit GP (Diassay, Rijswijk, The Netherlands)
Human papillomavirus genotyping test kit (PCR-RDB)-19 genotype (Daan Diagnostics, Burnaby,
Canada) and 1 variant
Human papillomavirus nucleic acid test kit (PCR-Fluorescence Probing) - 18 genotype (Daan
Diagnostics, Burnaby, Canada) and 2 variants
DR. HPV-27 Kit (Erba Diagnostics, Miami, FL)
Gel electrophoresis based full genotyping tests
BIOTYPAP Kit (Biotools, Nave, Spain)
PCR Human Papillomavirus Typing Set (Takara Bio Inc, Shiga, Japan)
Human papilloma virus genotyping (Genekam Biotechnology, Duisburg, Germany)
HPV High Risk Typing (Sacace, Como, Italy; Nuclear Laser Medicine S.R.L., Milano, Italia)
Real time PCR based full genotyping tests
PANA RealTyper HPV Kit (PANAGENE, Daejeon, South Korea)
AmpliSens HPV HCR genotype-FRT PCR kit (Federal State Institution of Science, Moscow, Russia; Ecoli
s.r.o., Bratislava, Slovak Republic)
Uterine Cervix Cancer of High-risk HPV Genotype Related Real Time PCR Kit (13 Types of
HPV Genotypes/2 channels) (Liferiver, Shanghai, China) and 1 variant
SuperFast HPV 12 Multiplex Real-time PCR Kit (Kogenebiotech, Seoul, Korea)
Uterine Cervix Cancer of High-risk HPV Genotype Related Real Time PCR Kit (13 Types of
HPV Genotypes/4 channels) (Liferiver, Shanghai, China) and 1 variant
HPV genotypes 14 Real-TM Quant (Sacace, Como, Italy; Nuclear Laser Medicine S.R.L., Milano, Italia)
HPV QUANT-15 (HPV 6,11,16,18,31,33,35,39,45,51,52,56,58,59,68) (DNA-Technology LLC,
Moscow, Russia) and 1 variant
Anyplex II HPV HR Detection (Seegene, Seoul, Korea) and 1 variant
RealBest DNA HPV HR Genotype (JSC "Vector-Best", Novosibirsk Region, Russia)
GeneProof Human Papillomavirus (HPV) PCR Kit (GeneProof, Brno, Czech Republic)
Medium or low density microarray based full genotyping tests

31
 PapilloCheck High-risk Test (Greiner Bio-One, Frickenhausen, Germany) and 1 variant
Clart HPV 2 - Papillomavirus Clinical Arrays (Genomica, Coslada, Spain)
21 HPV GenoArray Diagnostic Kit (Hybribio, Honk Kong, China) and 1 variant
GeneTrack HPV DNA Chip (Genomic Tree, Daejeon, South Korea)
GeneSQUARE HPV Microarray (Kurabo Industries, Osaka, Japan)
Infiniti HPV Genotyping Test (AutoGenomics, Carlsbad, CA) and 2 variants
PANArray HPV Genotyping Chip (PANAGENE, Daejeon, South Korea)
HPVDNAChip (Biomedlab, Seoul, South Korea)
GG HPV Genotyping Chip (GoodGene, Seoul, South Korea)
BMT HPV 9G DNA Kit (Biometrix Technology, Chuncheon, Korea)
BMT HPV Genotyping 9G Membrane Kit (Biometrix Technology, Chuncheon, Korea)
ProDect Chip HPV Typing kit (Bcs Biotech, Cagliari, Italy)
HPV type 3.5 LCD-Array Kit (Chipron, Berlin, Germany)
Decipher HPV23genotyping DNA chip (Yaneng Bioscience, Shenzhen, China)
MyHPV Chip (Mygene Co., Seoul, Korea)
EUROArray HPV (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany)
Microsphere beads based full genotyping tests
Multiplex HPV Genotyping Kit (DiaMex, Heidelberg, Germany)
LMNX Genotyping kit GP HR (Diassay, Rijswijk, The Netherlands)
PapType HR HPV detection and genotyping (Genera Biosystems, Melbourne, Australia)
GENOSEARCH-HPV31 (Medical & Biological Laboratories, Nagoya, Japan)
Tellgenplex High-risk HPV Genotyping Panel (Tellgen, Shanghai, China) and 1 variant
IntelliPlex HPV DNA Genotyping Kit (PlexBio, Taipei, Taiwan)
GeneFinder HPV PCR Kit + GeneFinder HPV Liquid Beads MicroArray Genotyping Kit (Infopia,
Gyunggi-do, Korea)
HPV 27 Genotyping Test Kit (Tellgen Corporation, Shanghai, China)
Mebgen HPV kit (Medical & Biological Laboratories, Nagoya, Japan)
Capillary electrophoresis based full genotyping tests
f-HPV typing (Genomed Diagnostics, Wollerau, Switzerland; Molgentix, Barcelona, Spain)
SureX HPV 25X Genotyping Kit (Health Gene Technologies Co. Ltd., Zhejiang, China)
HPV HR Detection Test (Trovagene, Inc., San Diego, CA)
Full genotyping tests based on PCR combined with matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry
HPV MassArray (BGI Americas Corporation, Cambridge, MA)
PapilloTyper (GeneMatrix Co., Seoul, Korea)
Full genotyping tests based on PCR combined with sequencing
GTPlex HPV PyroSeq Test (Genomic Tree, Daejeon, South Korea)
HPV Type-Detect 3.0 by Next Generation Sequencing (Reflex to HPV-16 Risk Assessment
Status) (Medical Diagnostic Laboratories, L.L.C., Hamilton, NJ) and 1 variant
HPV sign Genotyping Test (Qiagen Gaithersburg, Inc., MD, USA)
Full genotyping tests based on loop-mediated isothermal amplification and electrochemical DNA
chip
Clinichip HPV (Sekisui Medical, Tokyo, Japan)
706
707

32
708 Table 4: HPV DNA type- or group-specific genotyping tests present on the market in August 2015.

Strip, filter or microtiter-well hybridization based type- or group-specific genotyping tests

HPV TS 16 PCR-DEIA (Labo Bio-medical Products, Ev Rijswijk, The Netherlands) and 3 variants
GenoFlow STD array test kit (DiagCor Bioscience, Honk Kong, China)
RHA Kit HPV16 var (Labo Bio-medical Products, Ev Rijswijk, The Netherlands)
PCR Human Papillomavirus Detection Set (Takara Bio Inc, Shiga, Japan)
Gel electrophoresis based type- or group-specific genotyping tests
Human papillomavirus 16, 31, 33, 35, 35H, 58, 52, 67 (DNA-Technology LLC, Moscow, Russia) and 4
variants

HPV, Mixed Type, L1 Genes Primer Set (Maxim Biotech, Rockville, MD) and 7 variants
MPCR Kit for Human Papilloma Virus (Maxim Biotech, Rockville, MD)
Human Papilloma Virus (HPV-16/18) (High risk profile) (Genekam Biotechnology, Duisburg,
Germany) and 20 variants
HPV 16/18 PCR kit (BioCore, Seoul, Korea)
Real time PCR based type- or group-specific genotyping tests
AdvanSure HPV 16/18 real-time PCR (LGLS Diagnostics, Seoul, Korea)
Human papillomavirus 16, 18 (DNA-Technology LLC, Moscow, Russia)
HPV 16 & 18 Real Time PCR Kit (Liferiver, Shanghai, China) and 1 variant
Path-HPV16 (PrimerDesign, Southampton, UK) and 6 variants
Path-HPV16-standard (PrimerDesign, Southampton, UK) and 6 variants
HPV16-EASY (PrimerDesign, Southampton, UK) and 6 variants
AccuPower HPV16 & 18 Real-Time PCR Kit (Bioneer, Daejeon, Korea)
Human Papilloma Virus (HPV-16) (high risk profile) (Genekam Biotechnology, Duisburg, Germany) and
1 variant

Human papillomavirus high-oncogenic risk HPV (16, 31, 35), (33, 52, 58), (18, 39, 45, 59) (DNA-
Technology LLC, Moscow, Russia)
SuperFast HPV 16, 18, 6, 11 Multiplex Real-time PCR Kit (Kogenebiotech, Seoul, Korea) and 2 variants
Human PapillomaVirus Types 16 and 18 PCR Diagnostic Kit (Daan Diagnostics, Burnaby, Canada)
and 1 variant

High-risk Human Papilloma Virus (HR-HPV) PCR Kit (Daan Diagnostics, Burnaby, Canada)
RealLine HPV 16/18 (Str-format) (Bioron Diagnostics GmbH, Ludwigshafen, Germany) and 5 variants
and 5
RealLine HPV 16/18 (Fla-format) (Bioron Diagnostics GmbH, Ludwigshafen, Germany)
variants

HPV16/HPV18/HPV6+11 Multiplex Real Time PCR Kit (Liferiver, Shanghai, China)

HPV 16/18 Real-Time PCR (BioCore, Seoul, Korea)
5 Low-risk HPV Real-time PCR Kit (Hybribio, Honk Kong, China)
HPV QUANT-4 (HPV 6,11,16,18) (DNA-Technology LLC, Moscow, Russia)
HPV 6/11 Real-TM (Sacace, Como, Italy; Nuclear Laser Medicine S.R.L., Milano, Italia)
STD 12 multiplex PCR kit (GoodGene, Seoul, South Korea)
HPV-16 & HPV-18 by Multiplex Real-Time PCR (Medical Diagnostic Laboratories, L.L.C., Hamilton, NJ)
PapillomaFinder SMART 20 assay (PathoFinder, Maastricht, The Netherlands)
Fluorescence-based type- or group-specific genotyping tests
Human papillomavirus 16, 18 (DNA-Technology LLC, Moscow, Russia)
709
710

33
711 Table 5: hr-HPV E6/E7 mRNA tests present on the market in August 2015.

APTIMA HPV assay (Hologic, Madison, WI) and 1 variant

PreTect HPV-Proofer HV (NorChip, Klokkarstua, Norway)
NucliSENS EasyQ HPV (Biomerieux, Marcy l'Etoile, France)
QuantiVirus HPV E6/E7 mRNA for Cervical Cancer (DiaCarta LLC, Hayward, CA)
FLOWSCRIPT HPV E6/E7 assay kit (Enzo Life Sciences, Farmingdale, NY)
PreTect SEE (NorChip, Klokkarstua, Norway)
CervicGen HPV RT-qDX (Optipharm, Osong, Korea)
CervicGen hTERT RT-qDX (Optipharm, Osong, Korea)
QuantiVirus HPV E6/E7 mRNA for Head-Neck Cancer (DiaCarta LLC, Hayward, CA)
712
713

34
714 Table 6: in situ hybridization DNA and mRNA based HPV tests present on the market in August

715 2015.

in situ hybridization DNA based HPV tests

INFORM HPV III Family 16 Probe (B) (Ventana, Tucson, AZ) and 1 variant
GenPoint HPV DNA Probe Cocktail, Biotinylated (Dako, Glostrup, Denmark)
Wide Spectrum HPV DNA Probe Cocktail, Biotinylated (Dako, Glostrup, Denmark)
HPV Types 6/11 Biotinylated DNA Probe (Dako, Glostrup, Denmark)
ZytoFast HPV type 16/18 Probe (Biotin-labeled) (ZytoVision, Bremerhaven, Germany) and 2 variants
ZytoFast HPV type 16/18 Probe (Digoxigenin-labeled) (ZytoVision, Bremerhaven, Germany) and 2 variants
ZytoFast HPV type 16/18/31/33/35 Probe (ZytoVision, Bremerhaven, Germany) and 3 variants
HPV Screening (HPV6/11/16/18/31/33) (PanPath, Amsterdam, The Netherlands)
HPV type 16/18, labelled DNA probe (BIO) (PanPath, Amsterdam, The Netherlands) and 8 variants
HPV type 16/18, labelled DNA probe (DIG) (PanPath, Amsterdam, The Netherlands) and 8 variants
HPV In Situ Hybridization/Detection Kit (Types 6/11/16/18/31/33) (Maxim Biotech, Rockville, MD) and
3 variants

PATHO-GENE AP-NBT/BCIP HPV in situ screening assay (Enzo Life Sciences, Farmingdale, NY)
PATHO-GENE AP-NBT/BCIP HPV in situ typing assay (types 6/11, 16/18 and 31/33/51) (Enzo
Life Sciences, Farmingdale, NY)
PATHO-GENE HRP-AEC HPV in situ typing assay (types 6/11, 16/18 and 31/33/51) (Enzo Life
Sciences, Farmingdale, NY)
PATHO-GENE HRP-DAB HPV in situ typing assay (types 6/11, 16/18 and 31/33/51) (Enzo Life
Sciences, Farmingdale, NY)
BIO-AP REMBRANDT HPV screening kit (PanPath, Amsterdam, The Netherlands)
BIO-HRP REMBRANDT HPV screening kit (PanPath, Amsterdam, The Netherlands)
DIG-AP REMBRANDT HPV screening kit (PanPath, Amsterdam, The Netherlands)
DIG-HRP REMBRANDT HPV screening kit (PanPath, Amsterdam, The Netherlands)
BIO-AP REMBRANDT HPV typing kit (PanPath, Amsterdam, The Netherlands)
BIO-HRP REMBRANDT HPV typing kit (PanPath, Amsterdam, The Netherlands)
DIG-AP REMBRANDT HPV typing kit (PanPath, Amsterdam, The Netherlands)
DIG-HRP REMBRANDT HPV typing kit (PanPath, Amsterdam, The Netherlands)
panHPV, labelled DNA probe (BIO) (PanPath, Amsterdam, The Netherlands)
panHPV, labelled DNA probe (DIG) (PanPath, Amsterdam, The Netherlands)
HPV14 Probe Kit (BioGenex, Fremont, CA) and 1 variant
Bond Ready-to-Use ISH HPV Probe (Subtypes 16, 18, 31, 33, 51) (Leica Biosystems, Newcastle,
UK) and 1 variant
in situ hybridization mRNA based HPV tests
HPV OncoTect E6,E7 mRNA Kit (IncellDx, El Camino Real Menlo Park, CA; Invirion Diagnostics, Oak
Brook, IL)
HPV E6, E7 mRNA Probe (IncellDx, El Camino Real Menlo Park, CA; Invirion Diagnostics, Oak Brook, IL)
716

717

718

35