performance of commercial reverse line blot assays for hpv

1

Performance of Commercial Reverse 1

Line Blot Assays for HPV Genotyping 2

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Running Title: Performance of HPV line blot assays 4

5

Martin Steinau1*, Juanita M Onyekwuluje1, Mariela Z Scarbrough 1, Elizabeth R Unger1, Joakim 6

Dillner2, Tiequn Zhou3‡ 7

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1 WHO Human Papillomavirus Laboratory Network Global Reference Laboratory, Chronic Viral 9

Diseases Branch, Division of High-Consequence Pathogens and, National Center for Emerging 10

and Zoonotic Infectious Diseases Pathology, Centers for Disease Control and Prevention, 11

Atlanta, GA 30333, U.S.A. 2WHO Human Papillomavirus Laboratory Network Global Reference 12

Laboratory, Region Skåne, Malmö and Departments of Laboratory Medicine, Medical 13

Epidemiology & Biostatistics, Karolinska Institute, Stockholm, Sweden. 14

3Quality, Safety and Standards Team, Immunizations, Vaccines and Biologicals Department, 15

World Health Organization, Geneva, Switzerland 16

17

18

Key Words: Human papillomavirus, HPV typing, reverse line-blot assay 19

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.06576-11 JCM Accepts, published online ahead of print on 22 February 2012

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* Corresponding Author: 20

Martin Steinau, PhD 21

Centers for Disease Control and Prevention 22

1600 Clifton Rd, Atlanta, GA 30333 23

Ph: +01 (404) 639-0561 24

Fax: +01 (404) 639-3540 25

Email: [email protected] 26

27

Word Count: 28

Abstract: 217 29

Text: 3202 30

Tables: 5 31

Figures: 2 32

Financial Disclosures: This work was supported by the WHO via a project funded by the Bill and 33

Melinda Gates Foundation 34

Disclaimers: The findings and conclusions in this report are those of the authors and do not 35

necessarily represent the views of the supporting agencies. ‡The author is a staff member of 36

the World Health Organization. The author alone is responsible for the views expressed in this 37

publication and they do not necessarily represent the decisions or policies of the World Health 38

Organization. 39


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Abstract 40

The performance of three line blot assays (LBA) - Linear Array HPV genotyping assay (LA; Roche 41

Diagnostics), INNO-LiPA HPV Genotyping Extra (LiPA; Innogenetics) and the Reverse 42

Hybridization assay (RH; Qiagen) was evaluated using quantitated whole genomic HPV plasmids 43

(types 6, 11, 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, 59, 68b) as well as epidemiologic samples. In a 44

plasmid titration series LiPA and RH did not detect 50 international units (IU) of HPV 18 in the 45

presence of 5 x 104 or more IU HPV16. HPV DNA (1 - 6 types) in the plasmid challenges at 50 46

(IU) or genome equivalents (GE) were identified with an accuracy of 99.9% by LA, 97.3% by LiPA 47

and 95.4% by RH with positive reproducibility of 99.8% (Kappa = 0.992), 88.2% (Kappa = 0.928) 48

and 88.1% (Kappa = 0.926) respectively. Two instances of mis-typing occured with LiPA. Of the 49

120 epidemiologic samples 76 were positive for high-risk types by LA, 90 by LiPA and 69 by RH 50

with a positive reproducibility of 87.3% (Kappa = 0.925), 83.9% (Kappa = 0.899) and 90.2% 51

(Kappa = 0.942) respectively. Although all the assays had good concordance in the clinical 52

samples, the greater accuracy and specificity in the plasmid panel suggest that the LA assay has 53

an advantage for internationally comparable genotyping studies. 54

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Introduction 56

The introduction of HPV vaccines has highlighted the need for standardized accurate HPV 57

genotyping assays to assure comparability of results in laboratories world-wide, both before 58

and after vaccine introduction. A large variety of assays, using both in-house methods and 59

commercial kits, are available for HPV detection and typing (6), and more can be anticipated. 60

International proficiency testing organized by the WHO HPV LabNet has documented significant 61

differences in sensitivity, specificity and reproducibility between different laboratories using a 62

variety of testing platforms (2). Variation in performance for laboratories using the same assay 63

implicates laboratory practice; however the results also indicated differences between assays. 64

The WHO HPV LabNet has further recognized the need for HPV typing assays that can easily be 65

standardized, require minimal equipment for assay performance and interpretation, and which 66

can be used in a variety of laboratory settings. Following its meeting on the standardization of 67

HPV assays and the role of WHO HPV LabNet in supporting vaccine introduction (WHO/HQ, 68

Geneva, Switzerland, 23-25 January 2008, Ref: 69

http://www.who.int/biologicals/publications/meetings/areas/vaccines/human_ papillomavirus 70

/HPV%20Jan%20meeting%20report_20080909%20_Clean_.pdf) it was agreed that commercial 71

assay kits should be evaluated in collaborative studies for proficiency. 72

Reverse line blot assays (LBA), based on consensus amplification of conserved regions of HPV 73

followed by hybridization to type specific probes on line blot strips, have been widely used. 74

Available LBAs differ in the primer set (1,3,4) used in the amplification phase and in number and 75

sequence of the detection probes. Beyond thermocyclers for target amplifications, only 76

temperature control water baths and visual inspection are required to carry out these assays. 77


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Therefore the LBA platforms have the potential to meet the needs of the WHO for high 78

performance genotyping that does not involve expensive equipment. 79

Studies have been conducted previously to compare and evaluate performance of these HPV 80

typing assays. Typically, these were restricted to DNA extracts from patient-collected anogenital 81

specimens, which cannot be validated independently and limit the analysis to relative 82

comparisons and assessments of type prevalence found by the individual assays (5,7,10). The 83

use of plasmid standards offers clear advantages for more objective evaluations beyond that 84

level. Wittingly, cloned, full-length HPV genomic DNA provides complete control over genotype 85

identity as well as copy number input and eliminates the need for a gold standard test. 86

We selected three commercial LBAs using different primer sets and subjected them to a 87

number of different test samples. These included plasmid standards as well as patient extracts 88

from different sources. The objective was a comparison of these kits’ performance with the 89

potential needs of the WHO in mind. Specifically, assay sensitivity, specificity and 90

reproducibility were assessed. 91

92

Materials and Methods 93

Study Design: HPV plasmids were used to prepare test samples assessing the assays’ 94

performance parameters. Human genomic DNA was added as carrier diluents to a final 95

concentration of 1 ng/µL in each plasmid preparation to simulate the situation in actual 96

samples. For the evaluation of competition between HPV types with unequal copy numbers a 97

titration series containing a constant 50 IU of HPV18 DNA and different numbers of HPV16 IU in 98

10-fold increments from 5 x 100 to 5 x 106 was prepared. To evaluate detection of HPV at the 99


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desired level of sensitivity – 50 IU or GE per assay, the final concentration of each type was 100

adjusted so that 50 IU (or GE) would be assayed. Samples containing multiple types [from two 101

to six of the 15 types in different combinations] were prepared in order to evaluate impact of 102

multiple types on assay performance (see Table 1). Two additional control samples containing 103

no template DNA and one with only Human genomic DNA (Roche Diagnostics) were also 104

prepared. Since sample source and collection media can influence the performance of 105

molecular assays, particularly those based on PCR technology, we included extracts of cervical 106

cells in specimen transport media (STM) and archived formalin fixed paraffin embedded (FFPE) 107

tissue in the test panel. These samples are described below. In particular, FFPE extracts are 108

known to be challenging for assays with large amplicons, and the LBAs could perform 109

differently 110

The final testing panel included 66 plasmid challenge samples (7 titration, 15 individual, 18 111

multiple, , each in duplicate), 60 epidemiologic samples and 2 negative controls. Six replicate 112

aliquots of the testing panel were prepared to provide duplicate testing on each of the three 113

LBA platforms. All DNA samples were randomly coded by a member of the laboratory not 114

involved in testing disguising the origin of the sample, expected HPV status and types. Two 115

technologists independently tested a panel on each of the platforms and interpreted the 116

results. Prior to initiating testing, each technologist successfully completed proficiency testing 117

on each assay platform; correctly identifying one to five HPV types in 10 unknown samples with 118

at least 80% accuracy. 119

Each assay used a 10 µL DNA aliquot per PCR reaction. Results for all possible HPV types were 120

entered into MS Access database tables. A sample was termed HPV positive when at least one 121


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type was detected, HPV negative if none of the 15 types but the genomic control was positive 122

and inadequate if neither HPV nor control were detected. Testing was completed over a period 123

of two weeks. The order in which each technologist used each platform varied. 124

125

Plasmid samples: Full length clones of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 126

59, 68 in plasmid vectors (100 ng in TE buffer) were provided by the WHO HPV Global 127

Reference Laboratory (Region Skåne, Malmö, Sweden). Each plasmid was diluted to 5 x 104 128

genome equivalents per mL in 200 μg/mL yeast tRNA solution (Invitrogen, Carlsbad, CA). 129

Number of genomic copies for HPV16 and 18 had been previously standardized by WHO and 130

was measured in international units (IU) accordingly. Copy numbers of other types were 131

calculated from the molecular mass and concentration as Genome Equivalents (GE). Human 132

genomic DNA used as background for HPV plasmid samples was obtained from Roche 133

Diagnostics, (Indianapolis, IN and diluted to 10 ng/μl in 0.1 mM TE. 134

135

Epidemiological Samples: Residual DNA extracts were retrieved from anonymous archived 136

epidemiologic samples from studies of HPV, either populations with high HPV prevalence or 137

samples of HPV-associated cancers. These were randomly selected without knowledge of prior 138

HPV results to include each 30 DNA extracts from cervical cells in Digene STM (Qiagen, Valencia, 139

CA, USA) and 30 from archived FFPE cancer tissues. 140

141

HPV Genotyping: The selection of kits to be evaluated was based on the following criteria: (a) 142

ability to detect and individually identify all or the majority of the thirteen high risk HPV types 143


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16, 18, 31, 33, 35, 39, 51, 52, 56, 58, 59, 68b and the two low risk types 6 and 11, (b) no 144

requirment for expensive instrumentation to perform and interpret assay, and (c) commercial 145

availability in the US. Meeting most of these criteria, the Linear Array (LA) HPV Genotyping 146

Assay (Roche Diagnostics, Indianapolis, IN), INNO-LiPA HPV Genotyping Extra (Innogenetics, 147

Gent, Belgium) and the RH Probe Assay (Qiagen, Valencia, CA) were chosen. All three tests are 148

based on general HPV amplification via a low stringency PCR amplification of the L1 region and 149

subsequent detection via reverse line blot assay with type specific probes. Technical details are 150

compared in Table 2. 151

The LA uses PGMY 09/11 consensus primers and identifies a total of 37 individual types. The 152

test was performed according to the manufacturer’s specification, except that 40 μl DNase free 153

water was added with the 10 μl template DNA to fill to the required volume. Hybridization and 154

washing steps of the reverse line blot assay were done automatically with Beeblot instruments 155

(Bee Robotics, Caernarton Gwynedd, UK). HPV52 is only detected by an “XR” probe which 156

cross-hybridizes with HPV33, 35 and 58. In the presence of any of these three types, HPV52 157

cannot be identified unequivocally. 158

LiPA applies SPF10 primers and includes probes to detect 29 HPV types. The assay was 159

performed in accordance with the manufacturer’s protocol using an AutoBlot 3000H (MedTec, 160

Buffalo, IL) for hybridization to the genotyping strips. Some types are defined by a single 161

positive probe on the genotyping strip (i.e. HPV6, 11, 16), but others are interpreted as a 162

combination of two to four probes (i.e. HPV18, 33, 58). While the manufacturer provides 163

interpretation of type detection, including “possible” types, only those that were detected 164

unequivocally were included in the analysis. 165


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The RH assay was not officially available in the US at the time this study was conducted and the 166

kits were kindly provided by Qiagen. The test utilizes the GP5+/6+ primers and line probes to 167

distinguish 18 high risk types; it does not detect HPV6 and 11. PCR amplification and line blot 168

detection were carried out as specified by Qiagen. Hybridization and washing of HPV 169

genotyping strips was achieved manually using a Gemini Twin shaking water bath (SciGene, 170

Sunnyvale, CA) and an aspiration system with 8-needle Stream Splitter (Art Robbins Instrument, 171

Sunnyvale, CA). 172

A water blank and SiHa DNA were included as negative and positive controls in every run with 173

each assay to monitor validity. The results from these controls were not included in the 174

analysis. 175

176

Analysis: The 15 HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 were 177

considered for the analysis. Some calculations (positive samples, accuracy, reproducibility) for 178

RH results were restricted to the 13 high-risk types as indicated. The two technologists’ results 179

for each sample and platform were counted as independent events. For plasmid samples, 180

accuracy was calculated as the percentage of correctly detected types (true positive and true 181

negative) from the total number of types assessed in all samples. [Total types assessed = 15 182

types per assay x 66 samples x 2 assays = 990; 858 if restricted to 13 hr types] 183

Results from STM and FFPE extracts were compared descriptively. Positive reproducibility (PR) 184

was calculated as the percentage of HPV types identified in both replicates among the total 185

number of types detected in either of the duplicate samples. Unweighted Cohen’s Kappa 186


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coefficients were calculated with SPSS 15.0 for Windows for all types assessed (positive and 187

negative) in the relevant sample set. 188

189

Results 190

Titration of HPV16 against 50 IU HPV18: Detection of HPV16, HPV18 and genomic control are 191

listed in Table 3 at all input concentrations of HPV16. When HPV 16 is present below 5 X 104 IU, 192

all three targets are detected by all assays. While LA results were robust to increasing IU of HPV 193

16, LiPA and RH failed to detect 50 IU HPV18 and at the highest input of HPV 16 IU failed to 194

detect the genomic control. The negative control containing only human gDNA gave expected 195

results with all assays as did the no template control (data not shown). 196

197

Detection of HPV plasmid standards: Table 4 lists false positive, false negative and inadequate 198

results by LBA among plasmid samples. Both technologist’s results are reported so number of 199

results are double the number of samples. In the single HPV plasmid challenges, accuracy of LA 200

was 29/30 (96.7%), LiPA was 27/30 (90.0%), and RH was 23/30 (76.7%). [RH detected 23/26 201

(88.5%), if HPV6 and 11 are excluded.] The challenges with multiple HPV plasmids included a 202

total of 61 HPVs. Of these, LA assaysidentified all 122 types correctly (100%), LiPA 100/122 203

(82.0%) and RH 83/122 (68.0%). If HPV6 and 11 were excluded, 83/108 (76.9%) types were 204

correctly found by RH. 205

Accuracy for all 15 types in the 66 plasmid samples was 99.9, 97.3 and 95.4% for LA, LiPA and 206

RH respectively. Restricted to the 13 high-risk types, RH’s accuracy was 96.7%. 207

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Epidemiologic samples: Results for STM and FFPE samples are combined as differences 209

between the two sample types were negligible for all LBAs. From the 120 test results derived 210

from each assay, the genomic control probe was positive in 116, 90 and 87 tests by LA, LiPA and 211

RH respectively. However results were adequate in 118, 118 and 111 by LA, LiPA and RH 212

respectively because some samples were positive for HPV but negative for the genomic control. 213

One or more HPV types were detected in 76, 90, and 69 samples by LA, LiPA and RH 214

respectively. Complete type-specific agreement amoung all three LBAs was noted for 81 215

samples (67.5%). Of these 42 were HPV positive and 39 HPV negative. An additional 24 216

samples (20%) had at least one type detected concurrently by all assays and one sample was 217

HPV positive in all assays but without type-agreement. 218

The overall detection of individual types was very similar for the three LBAs with the exception 219

of HPV52, which was only detected by LiPA in 9 cases (Figure 1). LA detected a total of 111 220

types (108 without 6 and 11) in 76 samples, LiPA detected 121 types (111 without 6 and 11) in 221

90 samples and RH found 97 types in 69 samples. Instances of type concordances and 222

discordances between the assays are illustrated in Figure 2. 223

224

Reproducibility: All test samples with the exception of the seven titration samples were 225

included in the reproducibility assessment. In the 33 test samples prepared with single or 226

multiple plasmids, expected results were for a total of 76 instances of HPV type detection (67 227

without HPV6 and 11). Differences in reproducibility between STM and FFPE samples, were not 228

significant and they were combined in Table 5. 229

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Discussion 231

Results from the plasmid standards revealed differences between the HPV typing assays. The 232

titration of HPV16 in different amounts against constant HPV18 template numbers highlighted 233

the robustness of the assays to competing types with different concentrations. Copy numbers 234

of more than 5 x 103 or a 100-fold higher template amount of HPV16 suppressed amplification 235

of HPV 18 in LiPA and RH PCRs. These observations are in line with assessments by van Doorn at 236

al. (9), who found that 100 copies of HPV18 were outcompeted by 1000-fold higher 237

concentration of HPV16. Only the LA was able to detect both types even at 100,000 times 238

higher HPV16 concentrations. The larger reaction volume (100 μl in LA vs 50 μl in the others) 239

might be responsible for the superior tolerance to competition. The extreme differences in 240

copy number are unlikely to occur in actual biologic samples, however pushing the technical 241

limits of the assays allowed for evaluation of robustness to a variety of challenges. All three 242

LBAs generally could detect 50 IU/GE of the single high risk HPV types. Only HPV68b was not 243

detected by either RH duplicate, which is not surprising since the LOD is stated as 105 viral 244

copies in the RH Detection Kit Handbook. The sporadic lack of reproducibility may indicate that 245

this input amount is in the range of the lowest limit of detection (LOD) for at least some types 246

(Table 3). 247

Significant deficiencies were seen in samples that included more than one type. LiPA failed to 248

detect 18% of the types included in the 36 multi-plasmid challenges. The 22 missed types 249

consisted exclusively of HPV39, 58, 59 and 69. In some instances types 39 and 68b were 250

identified as “possible” types due to the LiPA’s multi probe set (see Table 4). Nevertheless, both 251

HPV39 and 68b were also missed in other samples that were free of this ambiguity suggesting 252


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unequal amplification efficiency or competition. Disregarding HPV 6 and 11 the RH assay failed 253

to detect 20.5% of the types in this subset. Besides HPV68b, types 52, 59 and 39 were missed in 254

several instances.. A critical limitation of the RH assay might result from the large discrepancy in 255

detection sensitivities for different HPV types. According to the handbook LODs differ 25,000-256

fold ranging from four copies for HPV16 to 100,000 copies for HPV53 and 68. 257

Reproducibility of results for the plasmid challenges was greatest among the LA test, but 258

generally good in all assays. Among the results derived from the patient samples RH had the 259

highest positive reproducibility (90.2%), but the denominator was also lowered to 51 types 260

since HPV6 and 11 are not detected for this test. It was rather surprising that no significant 261

differences were found between STM and FFPE extracts as fractured and poor quality DNA from 262

archived tissues should favor shorter amplicon lengths as targeted by the SPF primers (8). The 263

sample size may not have been sufficient to allow differences in assay performance to be 264

identified. Generally, results from the patient samples were comparable between the assays 265

with at least partial agreement in 87.5%. Performance differences found with the plasmid 266

samples might reflect rather extreme situations which are not relevant for the majority of real 267

clinical specimens. 268

False positive results were a particular concern. Two instances were observed among LiPA 269

results in plasmid samples. In both cases the algorithm for identifying the expected type 270

(HPV18, 58) requires that more than probe hybridizes with the amplicon. As only one of the 271

required probes hybridized, another type (HPV39, 52) was falsely indicated. It is likely that 272

individual LiPA probes differ in their affinity to the same amplicon and generate an incomplete 273

band pattern at low target amounts which would consequently lead to attributing the result to 274


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an incorrect type. Detection of HPV52 by LiPA might be particularly vulnerable to this problem. 275

In the LiPA assay the HPV 52 amplicon hybridizes to a single probe. However that probe also 276

hybridizes to amplicons of five additional HPV types that are distinguished by combinations 277

with other probes. In this regard it is noteworthy that HPV52 was detected in 9 of the 15 types 278

exclusively found by LiPA among the patient samples (Figure 1) and two of three patient 279

samples exclusively HPV positive by LiPA had only type 52. It seems likely that at least some of 280

these cases are false positive. 281

Conversely, some HPV52 may have been missed by the LA as it has a similar shortcoming and 282

detects HPV52 only through the cross-binding XR probe. While this ambiguity occurred in 28 283

cases among the LA results only two of them tested in fact positive for HPV52 by LiPA. 284

Every laboratory test is also influenced by the accuracy of human handling. The samples 285

prepared from plasmid DNA yielded one inadequate result each, by LA and LiPA and the (single) 286

HPV types not detected in these results were also counted as “missed”. Although performed 287

with utmost care in a clinically certified laboratory, operator errors cannot be ruled out as a 288

cause and may have lowered the real tests’ performances. However this possible distortion 289

should be minimal. 290

Analysis for this comparison study was restricted to the most relevant high risk types as well 291

asHPV6 and 11. It should be considered however, that Linear array detected 50, LiPA 16 and RH 292

4 additional HPV types in the 60 patient samples. Depending on the number of types covered 293

by each assay, the scope of HPV detection will always be limited and does not directly allow an 294

“HPV negative” interpretation. 295


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For the majority of samples, HPV typing results will be very similar and comparable for the 296

three LBAs evaluated. In difficult situations such as multiple infections, low copy numbers or 297

large difference in viral copies, LA has an advantage. Some limitations of LiPA are due to its’ 298

multiple probe detection system. RH is disadvantaged by low sensitivity for some types, 299

particularly HPV53, 68 and 82. LA performed nearly perfect and is only hampered by the cross-300

reacting XR probe for HPV52 detection. 301


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8. Tan SE, Garland SM, Rumbold AR, and Tabrizi SN. 2010. Human papillomavirus 327

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336 337


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Tables and Figures: 338 339 Table 1. HPV types in the plasmid test samples. 340

Plasmid Titration HPV type (Copy number)

Plasmid Individual*

Plasmid Mix*

HPV 18 (5E01), HPV 16 (5E00) HPV 6 HPV16, HPV68b

HPV 18 (5E01), HPV 16 (5E01) HPV 11 HPV18, HPV59






No HPV HPV 39 HPV45, HPV16, HPV52

Water blank HPV 45 HPV18, HPV39, HPV59

HPV 51 HPV6, HPV35, HPV51

HPV 52 HPV33, HPV18, HPV56

HPV 56 HPV58, HPV16, HPV11, HPV45

HPV 58 HPV6, HPV59, HPV52, HPV39

HPV 59 HPV31, HPV33, HPV35, HPV58, HPV56

HPV 68b HPV18, HPV31, HPV11, HPV45, HPV68b

No HPV HPV52, HPV39, HPV45, HPV68b, HPV51

HPV6, HPV31, HPV35, HPV16, HPV56, HPV59

HPV18, HPV33, HPV39, HPV51, HPV58, HPV68b

*All plasmids in the individual and mixed samples were prepared to represent the equivalent of 341

50 IU or GE per test sample; sensitivity suggested by the WHO HPV LabNet. 342

343

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Table 2. Technical specification of commercial LBAs. 345

LA LiPA RH

Primer set PGMY 09/11 SPF10 GP5+/6+

HPV amplicon size 450 bp 65 bp 150 bp

PCR volume 100 μL 50 μL 50 μL

Hybridization Temp. 53°C 49°C 50°C

Detection signal Horseradish Peroxidase

AlkalinePhosphatase

Alkaline Phosphatase

Genomic control

Control amplicon size

β-Globin

268 bp

HLA-DPB1

280 bp

β-Globin

258 bp

LA = Linear Array HPV Genotyping Test 346

LiPA = Line Probe Assay 347

RH = Reverse Hybridization 348

349

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Table 3. Impact of Increasing HPV 16 IU on Detection of 50 IU HPV 18* 351

Input HPV 18 = 50 IU

H.s. DNA = 10 ng LA LiPA RH

Test 16 IU HPV16 HPV18 β-Glob HPV16 HPV18 hDNA HPV16 HPV18 gDNA

1st 5E+00 + + + + + + + + +

2nd 5E+00 + + + + + + + + +

1st 5E+01 + + + + + + + + +

2nd 5E+01 + + + + + + + + +

1st 5E+02 + + + + + + + + +

2nd 5E+02 + + + + + + + + +

1st 5E+03 + + + + + + + + +

2nd 5E+03 + + + + + + + + +

1st 5E+04 + + + + fp + + + +

2nd 5E+04 + + + + + + +

1st 5E+05 + + + + + +

2nd 5E+05 + + + + + + +

1st 5E+06 + + + + +

2nd 5E+06 + + + + +

1st 0 + + +

2nd 0 + + +

* Detected = +, Not detected = blank; fp= false positive result – HPV 39 detected instead of HPV 18 352

353


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Table 4. Incorrect Line Blot Assay results in HPV plasmid challenges¥. 354

No. of types (No. of samples)

LA LiPA RH

1 (n = 30) 59 (ia) 39 (ia), 58 (fp 52), 59 6‡, 6‡, 11‡, 11‡, 52, 68, 68 2 (n = 14) 58, 59, 59 6‡, 6‡, 11‡, 11‡, 56, 52

3 (n = 8) 39*, 39*, 68, 59 52, 52, 68, 68, 59, 6‡, 6‡,

39 4 (n = 4) 58 (fp 52), 59, 59 11‡, 11‡, 6‡, 6‡, 39, 52, 59

5 (n = 6) 39, 39, 68, 68, 68*, 68* 58, 58, 39, 39, 52, 52,

11‡, 11‡, 68, 68, 68, 68 6 (n = 4) 39*, 39*, 58, 68, 59, 59 68, 68, 6‡, 6‡, 59, 59

355

ia = inadequate, fp = false positive and falsely detected type 356

* detected as “possible types” 357

‡ probe not included in assay 358

¥Each entry reflects failue to detect a type by one of the technologists. 359

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Table 5. Positive reproducibility of Line Blot Assays for plasmid standards and extracts from 361

epidemiologic samples. 362

363

LA LiPA RH *

Plas

mid

DN

A

(n =

33)

Types to be detected 76 76 67

Discrepant types 1 9 8

Samples affected 1 6 8

Positive Reproducibility 98.7% 88.2% 88.1%

Kappa 0.992 0.928 0.926

Epid

emio

logi

c (n

= 6

0)

Pos. types (by either test) 63 66 51

Discrepant types 8 11 5

Samples affected 6 9 5

Positive Reproducibility 87.3% 83.3% 90.2%

Kappa 0.925 0.899 0.942 364

* for RH analysis types 6 and 11 were omitted and the total number of types adjusted. 365

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367

368

Figure 1. Type-specific detection by the three LBAs in the 120 epidemiologic samples. White 369

bars indicate results found by by LA, gray by LiPA and black by RH LBA. 370

371

0

5

10

15

20

25

30

35

40

45

Num

ber o

f tim

es d

etec

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372

373

374

Figure 2: Venn diagram illustrating instances of type specific concordance and discordance between 375

assays (restricted to 13 high risk types- 18, 18, 31,33,35,39,45,51,52,56,58,59 and 68). Numbers in 376

parentheses indicate total number of HPV types detected by each LBA. 377

378

379

380

381

382

383

LA (108)

RH (97)

LiPA (111)

82

4

4 10

12 7

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performance of commercial reverse line blot assays for hpv

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