WO2008086830A1

WO2008086830A1 - Method for detecting the presence of pathogens, and especially human papillomavirus (hpv), in a cervicovaginal sample

Info

Publication number: WO2008086830A1
Application number: PCT/EP2007/007626
Authority: WO
Inventors: Wilhelmus Johannes Gerardus Melchers
Original assignee: Stichting Louis Pasteur.
Priority date: 2007-01-18
Filing date: 2007-08-31
Publication date: 2008-07-24

Abstract

The present invention relates to a method for detecting the presence of pathogens, and especially human papillomavirus (HPV), in a cervicovaginal sample of a female individual. Specifically, the method according to the present invention comprises: 1) isolating nucleic acid from a dried fluid spot of said cervicovaginal sample adsorbed on a solid carrier to obtain isolated nucleic acids; and 2) detecting the presence of pathogen derived nucleic acids in said isolated nucleic acids. According to the present invention, said cervicovaginal sample is especially obtained through self sampling by said female individual. The present invention further relates to a kit of parts for use with the present methods.

Description

METHOD FOR DETECTING THE PRESENCE OF PATHOGENS, AND ESPECIALLY HUMAN PAPILLOMAVIRUS (HPV) , IN A CERVICOVAGINAL

SAMPLE

Description

The present invention relates to a method for detecting the presence of pathogens, and especially human papillomavirus (HPV) , in a cervicovaginal sample of a female individual. According to the present invention, said sample is especially obtained through self sampling by said female individual. The present invention further relates to a kit of parts for use with the present methods.

The clinical value of, for example, human papillomavirus (HPV) testing is increasingly recognized and especially with respect to early detection of (developing) cervicovaginal cancers.

If used as primary screening method, human papillomavirus (HPV) assessment has a higher sensitivity and a higher negative predictive value, but a lower specificity for the detection of a pre-invasive disease compared to cervical cytology.

In the United Sates, the Food and Drug Administration (FDA) has authorized high-risk human papillomavirus (hr-HPV) assessment in women aged 30 and older for primary screening, in addition to cytological screening, and for the triage of ASC-US smears.

The Dutch Pathology association (NVVP) has recently approved additional high-risk human papillomavirus (hr-HPV) testing in all follow-up smears after the detection of a first-time borderline or mild dysplasia (BMD) smear.

High-risk human papillomavirus (hr-HPV) testing has a beneficial effect in patient management. Most likely this beneficial effect will increase when high-risk human papillomavirus (hr-HPV) assessment replaces cytology screening as primary screening tool.

Improving the sensitivity of cervicovaginal cancer screening methods through the implementation of high-risk human papillomavirus (hr-HPV) testing can increase the success rate of population-based screening programs.

Maximizing the participation-rate would further improve this success. In the Dutch screening program, approximately 70% of the invited women are actually taking part. Tragically, half of the cervical carcinomas are diagnosed in the remaining group of non-responders . Cervical cancer incidence would decrease significantly if these non-responders can be reached, providing that they do participate.

Women do not respond or participate for various reasons; being afraid of the procedure or the possible diagnosis, having a too busy schedule, or general unawareness . Genital self-sampling could be an easy accessible, user-friendly and time saving alternative for the physician-based collection of cervicovaginal sample material.

Material from vaginal lavages or self-sampling brushes is highly representative for the cervical human papillomavirus (HPV) status. A study has been published in which 1.6% of the population-based screening non-responders with a valid and hr-HPV positive self-sample test, appeared to have a high-grade cervical intra epithelial lesion.

This is significantly higher than the high-grade CIN prevalence observed in a population-based randomized controlled trial for implementation of high risk human papillomavirus (hr-HPV) testing in cervical screening, i.e., the Dutch POBASCAM trial. These results not only show that women who do not participate in a well-organized screening program indeed have a higher risk of developing cervical cancer, but also that high risk human papillomavirus (hr-HPV) testing on self-sampled materials provides a promising opportunity to increase the efficiency of population-based screening programs worldwide .

Moreover, non-responding women do actually participate in self-sampling projects, leading to an increased participation rate of population based screening programs and thus a higher rate of success.

The vast majority of studies assessing cervicovaginal samples obtained by self-sampling have used methanol-buffered solutions or other liquid transport media in liquid based cytology (LBC) vials, for example PreservCyt® solution and ThinPrep®.

Since these cervicovaginal samples can be inflammable, hazardous, and/or infectious, careful handling is required. At least in The Netherlands, regular mailing of fluid self- sampled samples is not allowed because of potential

(bio) hazard. This severely hampers the introduction of liquid based cervicovaginal self-sampling methods for the detection of pathogens, and especially human papillomavirus (HPV) such as high risk human papillomavirus (hr-HPV) . Therefore, it is an object of the present invention to obviate at least some of the above problems associated with cervicovaginal self-sampling, and especially with respect to (early) screening for (potentially) cervicovaginal cancers. This object, amongst others, is met by a method for detecting the presence of a pathogen in a cervicovaginal sample of a female individual comprising: — isolating nucleic acid from a dried fluid spot of said cervicovaginal sample adsorbed on a solid carrier to obtain isolated nucleic acids; and

- detecting the presence of pathogen derived nucleic acids in said isolated nucleic acids.

According to the present method, detection of the pathogen is provided based on nucleic acid isolation from a dried fluid spot of said cervicovaginal sample adsorbed on a solid carrier. The term "dried fluid spot", as used herein, is defined as one or more areas on a solid carrier on which an isolated cervicovaginal sample is transferred and subsequently absorbed and dried. In other words, the cervicovaginal fluids/mucus obtained using standard sampling methods is transferred onto a solid carrier capable of absorbing said cervicovaginal fluids/mucus, in stead of into the prior art fluid, and subsequently dried to provide the dried fluid spot according to the present invention.

Compared to the prior art disclosing transferring the cervicovaginal sample into a fluid after sampling, the dried cervicovaginal material absorbed on the solid carrier is less, or even not, infectious and stable thereby providing the possibility to transfer the sample from the place of sampling to a remote test facility by, for example, mail. Further, since the dried cervicovaginal sample can be easily, safely and stably transferred, even over large distances, the actual place of sampling is almost not restricted and can be any place such as the home of the female individual . Considering the inherent intimate nature of cervicovaginal sampling, performing the sampling anonymously and easily at home is expected, amongst others, to greatly improve the participating rate of genital disease screening programs and especially the (early) detection of cervicovaginal (developing) cancers due to (high risk) human papillomavirus (HPV) infection.

Although adsorbing bodily fluids, such as blood or milk, on a solid carrier is known, the present invention is, amongst others, based on the surprising discovery that nucleic acid based detection methods can also be performed on dried fluid spots of cervicovaginal samples with a similar or even greater detection efficiency compared to the present cytological screening thereby providing an "true" alternative .

As already indicated, the present method is especially suitable for detection of human papillomavirus (HPV) , and preferably high risk human papillomavirus (hr-HPV) infection in cervicovaginal samples considering clinical implications of such infection.

Therefore, in a preferred embodiment, the present invention relates to a method for detecting the presence of human papillomavirus (HPV, and preferably high risk human papillomavirus (hr-HPV) , in a cervicovaginal sample of a female individual comprising:

— isolating nucleic acid from a dried fluid spot of said cervicovaginal sample adsorbed on a solid carrier to obtain isolated nucleic acids; and — detecting the presence of human papillomavirus

(HPV, and preferably high risk human papillomavirus (hr-HPV) , derived nucleic acids in said isolated nucleic acids.

In a preferred embodiment of the present invention, the solid carrier capable of absorbing a cervicovaginal sample is a filter paper.

Filter papers are readily available, can be easily handled, reliable, stable and light weight thereby providing an especially suitable, and preferred embodiment, of the present solid carrier.

Suitable, and preferred, examples of filter papers according to the present invention are S&S 903 filter available by Schleicher & Schull or FTA elute filter papers available by Whatmann.

According to one preferred aspect of the present invention, the present cervicovaginal sample is air dried after being adsorbed on the present solid carrier, preferably a filter paper.

Air drying provides an easy way to obtain a dried fluid spot which can be performed without additional (specialized) drying equipment. Examples of suitable drying times according to the present invention are from 5 to 60 minutes, preferably from 10 to 30 minutes such as 15 minutes.

According to a most preferred aspect of the present invention, the cervicovaginal sample is obtained through self-sampling by the female individual.

Though self-sampling, the cervicovaginal sample in the form of a dried fluid spot can be autonomously and anonymously obtained without any professional medical assistance .

According to the present invention, the cervicovaginal sample is obtained using a vaginal brush using standard and readily accepted sampling methods. After obtaining the sample, the vaginal brush is contacted with the solid carrier, preferably the filter paper, according to the present invention to transfer the sample. A dried fluid spot is obtained by subsequently drying the sample. According to a preferred embodiment of the present invention, detecting the presence of the pathogen, preferably human papillomavirus (HPV, most preferably high risk human papillomavirus (hr-HPV) comprises detecting pathogen derived DNA. DNA can be easily isolated using standard methods and allows for an array -of^* subsequent analysis methods for pathogen detection and/or characterization such as Southern Blotting, RFLP, SNIP, RT-PCR, fluorescence, mass spectrometry, hybridization, sequencing, surface plasmon resonance and/or micro-array analysis .

According to a preferred aspect of the pathogen detection according to the present invention the pathogen, preferably human papillomavirus (HPV) , most preferably high risk human papillomavirus (hr-HPV) , is detected by subjecting the isolated nucleic acids to nucleic acid amplification to obtain an amplificate, and detecting the presence of pathogen derived nucleic acids in said amplificate.

Examples of suitable amplification techniques according to the present invention are PCR and NASBA.

According to a most preferred aspect of the present invention, the present pathogen is human papillomavirus (HPV) and detecting of human papillomavirus (HPV) derived nucleic acids, preferably DNA, comprises genotyping, preferably in combination with amplification.

Considering the many explicit and implicit advantages of the present detection method as described above, especially with respect to self-sampling, the present invention also relates to a kit of parts for providing a dried fluid spot of a cervicovaginal sample by self-sampling comprising :

— a cervicovaginal brush;

— a solid carrier;

— instructions for use. In the above kit of parts, the solid carrier is preferably a filter paper as described above.

According to a most preferred aspect of the kit of parts according to the present invention, the kit further comprising an envelope for transferring the cervicovaginal sample as a dried fluid spot on the solid carrier to a test facility.

The present invention, according to a further aspect, also relates to the use of a dried fluid spot of a cervicovaginal sample, preferably a self-sample, absorbed on a solid carrier, preferably a filter paper, for detection of pathogens, preferably human papillomavirus (HPV) , most preferably high risk human papillomavirus (hr-HPV) . Below, the present invention will be further described in the examples relating to specific detailed preferred embodiments thereof.

Examples

Example 1

Introduction

High-risk human papillomavirus (HPV) testing in cervical cancer screening has a beneficial effect in patient management and can increase the success rate of population-based screening programs.

Women not participating in these programs have a higher risk of developing cervical cancer. The introduction of cervicovaginal self-sampling is expected to increase the participation rate and thereby a reduction of cancer incidence .

This example demonstrates the use of self-sampled genital smears applied to filter papers (Dried Fluid Spots) allowing, amongst others, easy storage and transport.

Fifty women provided self-sampled genital smear applied to a filter paper and these cervicovaginal samples were subsequently assessed for (probable) high-risk HPV genotypes by the HPV SPFlO Line Blot 25 assay.

The HPV results were compared to a cervical smear taken by a trained physician. Twenty-five (50%) of all self-obtained samples were positive for (probable) high-risk HPV. Of these samples 23 also tested positive in the physician obtained smear. Twenty-four of the 50 samples (48%) were negative to (probable) high-risk HPV genotypes in both self-collected and physician smear. The overall agreement between self- and physician obtained sample was 96% (k-value: 0.92) .

This example shows that HPV detection and genotyping in self-obtained genital samples that are subsequently applied to Dried Fluid Spots is very suitable alternative for cervicovaginal samples sampled under medical supervision.

The detection efficacy is in a high overall agreement with HPV detection and genotyping in physician-obtained cervical smear. Compared to other self-sampling devices the Dried Fluid Spot method is not dependent on liquid storage methods that are potentially inflammable, hazardous.

Material and Methods

Study design The female participants were recruited between May and September 2006 at the Department of Obstetrics and Gynaecology of the Radboud University Nijmegen Medical Centre.

The participants had initially been referred to the gynaecologist because of two subsequent smears indicating borderline or mild dysplasia (BMD) . They underwent a colposcopic examination and were followed using an expectative management, allowing spontaneous regression of the lesion.

In this expectative management study setting, the patients were cytologically assessed every 6 months for 2 years at our outpatient clinic. Smear specimens were collected using the Cervex-brush® (Rovers Medical Devices B. V., Oss, The Netherlands), and processed using a liguid-based approach (Thinprep®, Cytyc corp . Boxborough MA, USA) that provides monolayer distributions for cytological assessment and the possibility of isolating DNA for HPV detection and genotyping assays. This method has received approval for clinical use from the U.S. FDA.

All patients were informed and the participants willing to participate were asked to sign a written informed consent.

A specific illustrated explanation was developed to instruct the women on how to obtain a genital self-sample. Briefly, a Viba-brush® (Rovers Medical Devices B. V., Oss, The Netherlands) was inserted 10 cm into the vagina and rotated 5 times.

Subsequently, the brush was applied to filter paper (Primagen Holding B. V., Amsterdam, The Netherlands). The Dried Fluid Spot was dried to air, placed in an envelope, and sent to the Department of Medical Microbiology for further processing and HPV assessment.

After self-sampling, a vaginal speculum was inserted and the physician obtained a cervical smear using a Cervex-brush® (Rovers Medical Devices B. V., Oss, The Netherlands) that was rinsed in a Thinprep® vial (Cytyc corp. Boxborough MA, USA) .

Regular liquid-based cytological (LBC) examination was performed at the Department of Pathology and 0.5 ml LBC medium was used for HPV assessment at the Department of Medical Microbiology.

In order to have the samples assessed anonymously and in a blinded approach, all self-obtained samples and cervical LBC samples were sent to the laboratory with a unique code that could only be encoded by the principal investigator.

Specimen preparation LBC

For isolation of DNA from cervical scrapes in liquid-based cytology medium, the MagNAPure LC Isolation station (Roche Diagnostics GmbH, Roche Applied Science,

Mannheim, Germany) was used.

500 μl of material was isolated using the MagNA Pure

LC Total Nucleic Acid Isolation Kit (Roche Diagnostics GmbH, Roche Molecular Biochemicals, Mannheim Germany), as described by the manufacturer. With each set of 28 cervical scrape samples 4 negative controls were included. Nucleic acid was resuspended in a final volume of 50μL; lOμL were used for

PCR.

Specimen preparation DFS

The dried fluid spots (DFS) were punched out of the filter paper using a sterilized perforator. DNA was isolated using the QIAGEN® DNeasy Tissue Kit (QIAGEN Inc, Valencia, CA, USA), as described by the manufacturer. Subsequently, HPV

DNA assessment was performed similar as for the liquid based cytology specimens, as described below.

SPFlO-INNO LiPA HPV detection and genotyping Broad-spectrum HPV DNA amplification was performed using a short PCR fragment assay (HPV SPFlO Line Blot 25, Labo Bio-medical products B. V. Rijswijk, The Netherlands). This assay amplifies a 65-bp fragment of the Ll open reading frame, and allows detection of at least 43 different HPV types .

The presence of HPV DNA was determined by hybridization of SPFlO amplimers to a mixture of general HPV probes recognizing a broad range of high-risk (hr) , low-risk (Ir) and possible hr-HPV genotypes in a microtiter plate format, as described previously.

Twenty-eight oligonucleotide probes recognizing 25 different types were tailed with poly(dT) and immobilized as parallel lines to membrane strips (Labo Bio-medical products B. V. Rijswijk, The Netherlands).

The HPV genotypes detectable are hr-HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68/73 and 2 probable hr-HPV types (53 and 66) . The HPV genotyping assay was performed as described previously. The LiPA strips were manually interpreted using the provided reference guide. Since HPV assessment in screening and triage is at present mainly solely based on (probable) high-risk genotypes, the comparison in example focuses on these genotypes.

As a quality control for the presence of DNA and absence of PCR inhibitors in the isolated material a beta-globin PCR was performed.

In all HPV 16, 18, 31, and/or 33 positive samples the viral load for the individual genotypes was assessed using real time PCR methods. The viral loads are indicated as copies per scrape.

Statistics All data were analyzed using SPSS version 12.0.1 for Windows. Agreement was measured by absolute agreement and Cohen's kappa statistics, a measure of the agreement between two methods that is in excess of that due to chance. Results

Fifty women were included in this example. The median age of the women was 35 years (range, 22 to 56) . All women were initially referred to the gynaecologist for 2 subsequent equivocal smears, i.e., borderline and mild dysplasia (BMD).

The LBC cervical smears taken by the physician showed normal cytology in 60% (30/50) of the cases, BMD cytology in 16/50 (32%) cases, and in 4/50 cases (8%) the smears appeared to be more severe than BMD. Of the 50 self-collected genital samples 25 (50%) tested positive for (probable) high-risk genotypes by the HPV SPFlO Line Blot 25. Of these 25 samples 23 also tested positive for (probable) hr-HPV in the cervical smear obtained by the physician. Twenty-four of the 50 samples (48%) were negative to (probable) high-risk HPV genotypes in both self-collected and physician smear. In 22 of the 50 (44%) cervical smears obtained by the physician hr-HPV types were detected, 21 cases also tested hr-HPV positive in the self-obtained sample. The overall agreement between self-sampling and the cervical smear taken by the physician for high-risk HPV was 92% (k-value: 0.84), see table I below.

Table I . High-risk HPV detection for corresponding genital self-sampled smears and MD obtained cervical smears

Self-sampling Agreement, κ-value and

MD-sampling

Positive Negative 95% CI

Positive 8 0

Normal Negative 3 19 cytology _Total 11 19 90%, 0.77 (0.53-1.02)^*

Positive 9 1

B_MD Negative 0 6

93.8%, 0.87 (0.63- cytology _Total 1.12)^**

Positive 4 0

>HMD NNeeggaatt:ive 0 0 cytology _Total 4 0 100%, 1.00 (1.00-1.0O)⁺

Overall Positive 21 1 Negative 3 25 Total 24 26 92% 0.84 (0. 69-0 . 99) strength of agreement considered good, very good, ⁺perfect

Taking both probable high-ri s k ( i . e . 53 or 66 ) and hr-HPV genotypes into account the overall agreement increased to 94 % ( k-value : 0 . 88 ) , see table I I below .

Table II. Probable high-risk and hr-HPV detection for corresponding genital self-sampled smears and MD obtained cervical smears

MD-sampling Self-sampling Agreement, κ-value and 95% CI Positive Negative ^ '

Positive 9 0

Normal Negative 2 19 cytology Total 1 1 19 93.3%, 0.85 (0.65-1.05)" __ .

Positive 10

BMD Negative 0 5 cytology Total 10 6 93.8%, 0.86 (0.60-1.12)"

Positive 4 0

>BMD Negative 0 0 cytology Total 4 0 100%, 1.00 (1.00-1.0O)⁺ __

Overall Positive 23 Negative 2 24

Total 25 25 94% 0.88 (0.75-1.01)" strength of agreement considered very good, ⁺perfect

The agreement of HPV DNA detection between the self-obtained and the physician obtained sample increased with the severity of the lesion detected in the LBC. For the probable high-risk and^' the high-risk genotypes together the agreement was 93%, 94% and 100% in the 30 smears with normal cytology, the 16 samples with BMD cytology and the 4 samples showing cytology more severe than BMD, respectively.

Regarding the defined hr-HPV genotypes the agreement was 90%, 94% and 100%, respectively.

The frequencies of the probable high-risk and high-risk HPV genotypes detected by both methods are summarized in table III below.

Table III. Distribution of HPV-genotypes in genital self- sampled smears and cervical smears taken by the physician

Oncogenic HPV Infection frequency Potential Genotype Self-sampling MD-sampling

16 5 5

18 2 2

31 5 4

33 0 2

35 0 0

M m 39 1 1 u i 45 0 0

51 3 1 X

52 3 4

56 2 2

58 1 1

59 2 1

68/73^a 2 2

Probable 53^D high-risk 66 aHPV SPFio Line Blot 25 does not distinguish between HPV 68 and HPV 73, since both types are detected by a single probe; "Oncogenic potential of HPV 53 is controversial.

Taking the samples of all 50 women into account, including those negative to probable high-risk and hr-HPV DNA, 40 samples (80%) were concordant, 7 (14%) were compatible, and 3 (6%) samples were discordant. Of the 40 concordant samples 24 showed no (probable) high-risk HPV DNA in both self- and physician obtained samples. The remaining 16 concordant samples were hr-HPV positive and showed identical genotypes in the two samples, see samples no. 1-16 in table IV below.

Table IV. Overview of the sample-sets containing at least one hr-HPV » genotype in either one or both samples including viral load for HPV 16, 18, 31, and/or 33.

HPV genotype Viral load (16, 18, 31, or 33)

(HPV SPFio Line Blot 25 genotypmg test) (copies/scrape)

.. MD-sampling Self MD Self MD sampling

Concordan 1.5710 8.1710

1 31 31 t 7 7

2 52 52 Concordan t

Concordan 2.7710 7.4710

3 18 18 t 7 6

4 Concordan

68/73 68/73 t

Concordan 2.0710 1.7710

5 18 18 t 6 4

6 56, 66 Concordan

56, 66 t

Concordan 5.9710 1.1710

7 16, 53 16, 53 t 5 5

Concordan 2.7710 3.5710

8 16 16 t 6 5

7.9710 1.6710

9 16 16 Concordan t 4 6

10 Concordan

58 58 t

11 Concordan

56 56 t

12 Concordan

39 39 t

13 59 Concordan

59 t

14 Concordan

52 52 t

15 68/73 68/73 Concordan t

16 Concordan

51 51 t

Compatibl 7.7710 9.6710

17 31 31, 52 e 4 2

Compatibl 5.0710 2.8710

18 16, 59, 66 16 e 4 5

Compatibl

19 51, 66 66 e

5.0710 1.2'10

Compatibl

20 16 16, 31 5 (HPV 4 (HPV LND e (HPV 31) 16) 16)

Compatibl LND

21 31 31, 33 LND 4 (Hpv e (HPV 31) (HPV 31) 33)

Compatibl

22 31, 52 52 e LND

23 Discordan

31 - t LND

24 - Discordan

51 t

25 33 Discordan

- LND t

LND : viral load not detectable The seven compatible sample-sets, i.e., showing one or more comparative HPV genotypes between the samples, were multiple infections when considering both samples. In 6 self-obtained and 5 MD-obtained samples a hr-HPV type was detected, see Table IV samples no. 17-22.

In 3 cases, the MD-obtained sample did contain a specific hr-HPV genotype that was found in the self-obtained sample, and in three other cases the self-obtained sample did not contain a hr-HPV type. In one compatible sample-set, no hr-HPV was detected in either one of the samples. In the three discordant cases a hr-HPV genotype was detected in either one of the samples, sample no. 23-25 in Table IV. In two samples the MD-smear did not contain a hr-HPV type in contrast to the self-obtained sample. Whereas in one sample-set only the MD-obtained sample contained a high-risk genotype.

The average beta-globin of the self-obtained samples was 4.5E+4 (±3.0E+4), whereas the average beta-globin of the samples taken by the physician was 6.4E+3 (±6.1E+3). This difference was statistically significant P<0.001 (Unpaired T-test) .

Table IV provides a summary of the 25 sample-sets that contained at least one high-risk HPV genotype in either one or both samples, 16 concordant samples, 6 compatible, and 3 discordant sample-sets. In case a sample contained HPV 16,

18, 31, and/or 33 the viral loads per scrape per genotype are given .

Although numbers of samples with a viral load were too small for proper statistical analysis, the loads show little variance between the self-obtained and the MD obtained sample in case the viral loads could be detected.

In five samples however, the viral load was too low for detection using the real-time PCR method and could thus not be detected. In four cases it concerned a genotype which was only detected in either one of the samples and in 1 case it concerned HPV 31 which was detected in both genital-self and MD obtained sample.

Discussion

The present example shows that HPV detection and genotyping in self-obtained genital samples that are subsequently applied to dried fluid spots (DFS) is possible. Moreover, this method shows a high overall agreement with HPV detection in physician-obtained cervical LBC.

All but three DFS hr-HPV positive samples were also hr-HPV positive in the MD-smear, whereas only one DFS hr-HPV negative sample appeared to be hr-HPV positive in the MD-smear. Leading to an overall agreement between the two methods of 92%, and 94% taking the high-risk and probable hr-HPV genotypes into account.

Also high concordance between specific HPV genotypes assessed in the self-sample and the cervical smear by the physician signifies that the samples self-obtained by the patients are representative for the genotypes that infect the cervix .

Moreover, the amount of beta-globin found was significantly higher in the self-obtained samples (T-test, P<0.001), implying that limited cell count and adequacy in self-obtained samples is no limitation.

The dried fluid spots used in the present method are neither (bio) hazardous nor inflammable; applying genital scrapes on these filter papers solves storage and transportation problems. Since this technique is also applicable to at-home self-sampling, it implies an incentive for decreasing non-compliance of the population-based screening . Especially women not participating due to fear might be persuaded to actually participate. Moreover, since self-sampling can be done by the patient and only the women who are persistently hr-HPV positive ought to be examined, less physicians and MD-time is needed, which will eventually reduce costs.

Conclusion

In conclusion, HPV detection and genotyping on self-sampled cervicovaginal samples is very well possible and the results are highly representative for the cervical HPV status .

The dried fluid spot technique is non-hazardous and the samples are allowed regular mailing. This suggests provides that the DFS method is applicable to at-home self-sampling in the population-based screening non-responders, for the introduction of a primary HPV-based cervical cancer screening, and for establishing cervical cancer screening programs in developing countries.

Example 2

Kits of parts comprising a FTA elute Whatmann filter paper, a vaginal brush, an envelope and instructions for use were sent to young females not suspected to have cervicovaginal abnormalities. As a control, also a prior art tube comprising a fixative fluid for cervicovaginal samples was sent additionally.

After self-sampling, the vaginal brush was contacted with the filter paper and, as a control, the vaginal brush was added to the tube comprising the fixative fluid.

After air drying of the filter paper, the filter paper was sent in the envelope to a test facility by regular mail . Also the tubes comprising the vaginal brush were collected for further analysis.

DNA was isolated from the dried fluid spots and amplified and genotyped as described above. As a control, the same procedure was followed for the tubes comprising the vaginal brush (at present, the "golden standard" for high risk human papillomavirus screening) .

The results are depicted in table V below. HPV test sure path indicates human papillomavirus detection in fixative and HPV test filter indicates human papillomavirus detection using dried fluid spots. DEIA indicates the presence (+) or absence (-) of human papillomavirus (HPV and

LiPA the human papillomavirus genotype detected.

As can be seen in table V, te results obtained for both detection and genotyping of human papillomavirus using dried fluid spots was superior over prior art fluid fixative testing. The filter test was able to identify two additional

HPV positive women (indicated in bold) tested negative in the prior art test.

Table V: A comparison between fluid and dried fluid spot human papillomavirus detection and genotyping

Claims

1. Method for detecting the presence of a pathogen in a cervicovaginal sample of a female individual comprising: — isolating nucleic acid from a dried fluid spot of said cervicovaginal sample adsorbed on a solid carrier to obtain isolated nucleic acids; and — detecting the presence of pathogen derived nucleic acids in said isolated nucleic acids.

2. Method according to claim 1, wherein said pathogen is human papillomavirus (HPV) .

3. Method according to claim 2, wherein said human papillomavirus (HPV) is a high risk human papillomavirus (hr- HPV) .

4. Method according to any of the claims 1 to 3, wherein said solid carrier is a filter paper.

5. Method according to any of the claims 1 to 4, wherein said cervicovaginal sample adsorbed on said solid carrier is air dried.

6. Method according to any of the claims 1 to 5, wherein said cervicovaginal sample is obtained through self- sampling by the female individual.

7. Method according to any of the claims 1 to 6, wherein said cervicovaginal sample is obtained using a vaginal brush.

8. Method according to any of the claims 1 to 7, wherein detecting the presence of said pathogen comprises detecting pathogen derived DNA.

9. Method according to any of the claims 1 to 8, wherein the presence of said pathogen is detected by subjecting the isolated nucleic acids to nucleic acid amplification to obtain an amplificate, and detecting the presence of pathogen derived nucleic acids in said amplificate.

10. Method according to any of the claims 2 to 9, wherein said pathogen is human papillomavirus (HPV) and detecting of human papillomavirus (HPV) derived nucleic acids comprises genotyping.

11. Kit of parts for providing a dried fluid spot of a cervicovaginal sample by self-sampling comprising:

— a cervicovaginal brush; - a solid carrier;

- instructions for use.

12. Kit according to claim 11, wherein said solid carrier is a filter paper.

13. Kit according to claim 11 or claim 12, further comprising an envelope for transferring the cervicovaginal sample as a dried fluid spot on the solid carrier to a test facility.

14. Use of a dried fluid spot of a cervicovaginal sample absorbed on a solid carrier for detection of pathogens .

15. Use according to claim 11, wherein said solid carrier is a filter paper.

16. Use according to claim 14 or claim 15, wherein said cervicovaginal sample is a self-sample.

17. Use according to any of the claims 14 to 16, wherein said pathogen is human papillomavirus (HPV) .