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Journal of Virological Methods 149 (2008) 163–166

Short communication

Detection of high-risk human papillomavirus by two molecular techniques:Hybrid capture and linear array

Jesus Chacon de Antonio ∗, Ana Fernandez-Olmos, Marıa Mercadillo,Marıa Luisa Mateos Lindemann, Fernando Baquero Mochales

Service of Microbiology, Ramon y Cajal University Hospital, Ctra. Colmenar, Km 9.1, 28034 Madrid, Spain

Received 18 April 2007; received in revised form 24 January 2008; accepted 24 January 2008Available online 6 March 2008

bstract

A number of human papillomaviruses (HPV) are the etiological agents of cervical cancer. The present study compared the performance of theybrid capture method with that of linear array for the detection of high-risk HPV in 218 cervical samples. For the linear array technique, theNA was extracted using two different procedures, one manual and the other automated. There was no difference in high-risk HPV (HR-HPV)

etectability between the two extraction procedures but the automated procedure had the advantages of simplicity, time and efficiency. There wasgreement in 199 (91.3%) of the results. The K value for the two assays was 0.81 indicative of “near perfect” agreement. Both methods, hybridapture and linear array, are sensitive options for detection of HPV in cervical samples. Linear array enables the identification of the genotyperesent in the sample and the presence of multiple infections.

2008 Elsevier B.V. All rights reserved.

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eywords: Hybrid capture; HR-HPV linear array

. Introduction

Human papillomaviruses represent one of the most com-on sexually transmitted infections in sexually active men andomen. Some of the approximately 100 HPV genotypes thatave been identified are potentially oncogenic, and are detectedn cervical cancer (IARC, 1995; Cogliano et al., 2005).

A number of techniques based on the detection of HPV (fun-amentally molecular detection of viral DNA) are available forhe screening of cervical cancer. These molecular techniquesnable the identification of a number of different HPV geno-ypes, including those considered to imply high oncogenic riskHR-HPV) (Munoz et al., 2003).

One of these techniques, currently available commerciallynd validated for use in the screening of HPV is the Hybrid Cap-ure II® test (DIGENE, Gaithersburg, USA). This test involves

ybrid capture and can detect the presence of viral DNA by initro formation of hybrids with specific RNA probes. It is ableo identify 13 genotypes of HR-HPV, including the set of poten-

∗ Corresponding author. Tel.: +34 91 3368787; fax: +34 91 3368809.E-mail address: jchacon.hrc@salud.madrid.org (J.C. de Antonio).

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166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2008.01.021

ially oncogenic types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56,8, 59 and 68) (Lorincz and Anthony, 2001). The test can alsoetect some low risk genotypes (6, 11, 42, 43 and 44).

Another technique available is the Linear Array® (Rocheiagnostics S.L., Barcelona, Spain). This is an array-based HPVenotyping test based on the amplification of the viral DNA byeans of the polymerase chain reaction (PCR) technique and

ybridization of nucleic acid to specific HPV probes. This testdentifies 37 HPV DNA genotypes in cervical samples (Gravittt al., 2000), including the 13 high-risk genotypes detected byhe hybrid capture technique (16, 18, 31, 33, 35, 39, 45, 51, 52,6, 58, 59 and 68), together with other possible high-risk, inde-erminate nature, and low-risk genotypes (6, 11, 26, 40, 42, 53,4, 55, 61, 62, 64, 66, 67, 69,70, 71, 72, 73, 81, 82, 83, 84, IS39nd CP6108).

The aim of the present study was to compare two availableommercially tests for the detection of HR-HPV in cervical sam-les: the Hybrid Capture II® HPV DNA test and the Linearrray® HPV test.

. Material and methods

All assays were performed by trained laboratory technicians.

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.1. Clinical specimens

The study comprised a total of 218 cervical scrape sam-les from 218 women who attended the gynaecology outpatientlinic of Ramon y Cajal Hospital in Madrid (Spain). The sam-les were collected with cervical brushes (Cervix-Brush®), andtored in PreservCyt® medium. The HPV DNA present in theamples was extracted using two different techniques (manualnd automatic procedures) in order to compare both extractionethods.

.2. Hybrid capture method

The Hybrid Capture II® HPV DNA detection test uses a probeo detect the presence of HR-HPV, and another probe to detecthe presence of low-risk virus (LR-HPV). The amplification sig-al is based on the production of DNA/RNA hybrids that arenalyzed via chemiluminescence. The results are expressed ashe ratio RLU/CO (RLU: relative light unit, CO: cutoff or lim-ting value). The test was considered positive when the RLUsere equal to or greater than the mean of three internal controls,

quivalent to 1 pg/ml of HPV DNA (RLU/CO ≥1). The lightntensity emitted by the positive sample is directly proportionalo the amount of HPV DNA present in the sample, and is takens a semiquantitative estimation of the viral load in the sample.he technique was carried out following the instructions of theanufacturer.

.3. Linear array

The Linear Array® HPV genotyping test is based on fourain processes: preparation of the samples, amplification of

he DNA by means of primers for HPV, hybridization of themplified products with oligonucleotide probes, and detectionf the amplified products fixed to the probes via colourimetricetermination.

Genotyping with the linear array uses biotinylated primers toefine a sequence of nucleotides in the polymorphic L1 regionf the HPV genome, with a length of approximately 450 baseairs. An additional primer targeted to the �-globin as internalontrol is also used and is included in the kit.

For this linear array technique, the DNA was extracted andsolated using two different procedures—one manual and thether automatic (Stevens et al., 2006).

.3.1. DNA extraction methodsOnly manual extraction was used for the first 73 samples.

oth extraction procedures, manual and automatic, were usedor the subsequent 100 samples. Only the automatic procedureas used for the remaining 45 samples (a total of 218 sam-les). This decision was based on the absence of differencesn HR-HPV detectability between the two procedures, and onhe advantages of the automatic extraction procedure. Positive

nd negative controls were included in all assays. The automaticrocedure COBAS AmpliPrep Instrument (Roche DiagnosticsmbH, D-68298 Mannheim, Germany) purifies RNA or DNA

argets for PCR in six steps: loading the sealed sample inputh1

ical Methods 149 (2008) 163–166

ubes, lysis and hybridization, capture, washing, resuspension,ransfer of prepared samples to output tubes and then their returno their original positions in the rack.

In the manual procedure, the HPV DNA was extracted from250-�l aliquot of sample, using the Roche AmpliLute liq-

id media extraction (EXTRN) kit with the Qiagen QIAampinElute Columns and Qiagen QIAvac 24 Plus vacuum sys-

em (Roche Molecular Systems, Inc. Branchburg, NJ, USA)ccording to the instructions of the manufacturer.

In the automatic procedure, the EXTRN kit (Roche Diagnos-ics S.L., E-08006 Barcelona, Spain) was also used. The HPVNA was extracted from an 800-�l aliquot of sample added to50 �l of tissue lysis buffer using the COBAS Ampliprep Instru-ent, where a multi-reagent cassette modification procedure of

he total nucleic acid isolation kit was employed. We used neu-ral elution buffer, RNasa-free water (included in the EXTRNit) instead of the acid elution buffer which was in the totalucleic acid isolation elution buffer container, and added car-ier RNA and diluent (tris–hybrid capture buffer) to the emptyeneral purpose vial.

.3.2. HPV genotypingHPV genotyping was performed by two kits: linear array

PV genotyping test (PCR amplification and hybridization) andinear array detection kit (detection).

PCR amplification: each 100 �l reaction consisted of 50 �lorking master mix and 50 �l of DNA sample. Amplificationas performed in an Applied Biosystems GeneAmp PCR Sys-

em 9700 using the recommended parameters: 50 ◦C for 2 min,5 ◦C for 9 min and 40 cycles of 95 ◦C for 30 s, 55 ◦C for 1 min,2 ◦C for 1 min and finally, at 72 ◦C for 5 min before holding itndefinitely at 72 ◦C.

Hybridization to the oligonucleotide probe: 100 �l of DSdenaturing solution) was added to the PCR product. In all thenalyzed samples, determination of the genotype was carriedut using an automatized procedure based on the recommendedrotocol but with the modification of using a 100-�l aliquotf the denatured amplicons rather than a 75-�l aliquot. Theenatured amplicons were hybridized on to the strip containingpecific probes for 37 HPV genotypes and �-globin referenceines before undergoing stringent washes. This results in thePV specific probes being bound to a nylon membrane on which

he hybridization occurs.Colourimetric determination: a blue coloured complex pre-

ipitates at the probe positions where hybridization occurs.hen, the linear array HPV genotyping strip is read visuallyy comparing the pattern of blue lines to the linear array HPVenotyping test reference guide.

The presence of �-globin was observed in all the samplesested thus demonstrating the adequate integrity of the sample,igh efficiency of DNA extraction and amplification.

. Results

In 99 of the 218 samples studied, HR-HPV was detected byybrid capture and the sample viral load readings were betweenand 4251 pg/ml; of these 99 samples, 94 revealed HR-HPV

J.C. de Antonio et al. / Journal of Virological Methods 149 (2008) 163–166 165

Table 1Results of the detection of HPV using the two techniques studied (Hybrid Capture II® and Linear Array®)

Hybrid capture positive Hybrid capture negative Hybrid capture total

LA positive 94 (43.1%) 14 (6.4%) 108 (49.5%)LA negative 5 (2.2%) 105 (48.1%) 110 (50.4%)

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A total 99 (45.4%)

ybrid Capture: Hybrid Capture®; Kappa index: 0.817; LA: Linear Array®.

enotypes with the linear array test, using either of the twoxtraction procedures with discrepancies in the remaining fiveamples (2.3%), in which no HR types were detected. Samplesith discrepant results were not repeated. In these five sam-les, the viral load readings were between 1 and 27 pg/ml. Inne of the samples, with a viral load of 1 pg/ml, an LR-HPVas detected by linear array, while in the remaining four nei-

her HR-HPV nor LR-HPV was detected by linear array. Bothxtraction procedures were used in these five samples.

In 108 (49.5%) of total 218 samples HR-HPV was detected byinear array. In 94 of these, HR-HPV was identified by hybridapture, with discrepant results in the remaining 14 samples6.4%), in which no HR types were detected. Interestingly, in 7f these 14 samples, the bands of the HR-HPV genotypes onlyppeared with manual extraction; in 4 of the 14 samples theyppeared with manual extraction, although automatic extractionas also carried out. Lastly, in 3 of the 14 samples the genotypesere recorded by both extractions. Of note is the fact that in the5 samples in which only automatic DNA extraction was carriedut, no discrepancies were recorded between HR-HPV detectionia hybrid capture and linear array.

On the other hand, when the two extraction methods weresed in 100 samples, four discrepancies in HR-HPV detectabilityere found. HR-HPV was found only with manual extraction.To summarize, there were discrepancies in 19 (8.7%) of

he 218 samples analyzed, and agreement of the results inhe remaining 199 samples (91.3%). The concordance levelsetween both methods were established with the kappa index,hich yielded a numerical value of 0.817. The results are shown

n Table 1.

. Discussion

Several studies have compared the two techniques used mostommonly, hybrid capture and linear array, for the detection ofPV in cervical samples.The results of these studies differ greatly, since they depend

n the type of PCR employed, the primers included, the lengthf the amplified DNA fragment, the detection method in general,nd the conditions of the test.

Some investigators have reported agreement of the resultsbtained with the two techniques, hybrid capture and linearrray, in 90% of cases in women with abnormal Pap smear resultsMonsonegro et al., 2005). However, other studies have found

nalogous results with the two techniques used for the detectionf high grade lesions, but not in women with normal cytologicalndings (27% prevalence of HPV DNA via hybrid capture andnly 13% with linear array) (Schneede et al., 2001). Therefore,

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119 (54.5%) 218 (100%)

n view of the disparity of data in the literature, it is impor-ant to establish a technique that offers reliable results, is easyo perform and with a sensitivity and negative predictive valuelose to 100%. The more automatized the technique used forhe detection of HPV DNA, the closer we will come to achieveur objective in the prevention of cervical cancer. In this study,he results obtained by hybrid capture and linear array coin-ided in 199 samples (91.3%), yielding a kappa index of 0.817,hich according to Cohen (1960) is indicative of “near perfect”

greement.The hybrid capture technique identifies whether the HPV

resent in the sample is of low- or high-risk, but it does not deter-ine the genotype or the presence of multiple infections. This

s the only technique currently approved by the Food and Drugdministration (FDA). It is generally considered the most ade-uate procedure for the screening of cervical cancer in womenged 30 years or older, in combination with cytology (Wright etl., 2004). Although hybrid capture is very sensitive, it dependsn the cell content of the samples, occasionally yielding falseositive readings with low RLU/CO values, as well as possi-le false negative readings and cross-contaminations betweenigh- and low-risk types (Yamazaki et al., 2001). This is per-aps more evident in multiple infections and in women withormal cytological findings.

Some commercially available methods enable the precisedentification of the HPV genotypes present in the samples. Theinear array technique is a standardized, consistent and rapidechnique for the detection of 37 HPV genotypes. In our study,

manual and an automatic extraction procedure were used,ith few discrepancies between the two methods. The auto-atic extraction of DNA is superior in terms of simplicity, time

nd efficiency in the laboratory—practically eliminating the riskf sample contamination. Since sporadic contamination is notasily identifiable with manual extraction, the automatic extrac-ion procedure is more advisable for routine and widespreadaboratory use.

. Conclusions

The two tests used in this study for the detection of HPV inervical samples – hybrid capture and linear array – are use-ul and reliable tests to detect the presence of HPV cervicalnfection. It should be pointed out that HPV genotyping withhe linear array offers a series of advantages such as identifica-

ion of the genotype present in the sample, making it possibleo identify persistent infection – this being an important factorn the progression of precancerous cervical lesions – and theresence of multiple infections. Other additional advantages are

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hat it allows comparison of the genotypes obtained in specificetection programs before and after introduction of the vaccineKoutsky et al., 2002), and the facilitation of epidemiologicalesearch into the natural history of HPV infections.

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