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Correspondence |
1 Faculty of Medicine, Makerere University, Kampala, Uganda
2 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden
3 International Agency for Research on Cancer, Lyon, France
4 DDL Diagnostic Laboratory, Voorburg, the Netherlands
5 School of Public Health, Makerere University, Kampala, Uganda
6 Uganda Virus Research Institute, Entebbe, Uganda
7 Cancer Registry of Norway, Oslo, Norway
8 Samfundet Folkhalsan, Helsinki, Finland
Correspondence
Cecily Banura (c/o Elisabete Weiderpass)
(Elisabete.Weiderpass.Vainio{at}ki.se)
Testing for high-risk human papillomavirus (HPV) types has been recommended for primary cervical cancer screening, as well as for management of women with low-grade cervical cytological abnormalities (IARC, 2005). New, inexpensive screening methods for rapid identification of high-risk HPV types are under evaluation (Lorincz, 2006) and may facilitate the use of HPV testing in low-resource countries.
Cervical exfoliated cells for HPV testing are usually collected in PBS, which is cheap, but requires constant refrigeration, or in other liquid-based preservation media (ThinPrep Solution; Cytyc), which can be stored at room temperature, but are expensive and flammable. Filter paper has been widely used for collecting dry blood spots to screen for various infectious agents (Beebe & Briggs, 1990; Gupta et al., 1992; Kain & Lanar, 1991) and metabolic and genetic diseases (Bickel et al., 1981; Garrick et al., 1973; Maeda et al., 1985). Paper samples would greatly facilitate the collection, storage and transport of exfoliated cervical cells in low-resource countries. Kailash et al. (2002) reported similar detection rates of HPV16 in paper and PBS samples among 100 women with cervical dysplasia and carcinoma in India. To further evaluate the issue, we compared the detection of 25 HPV types in paper and PBS samples among young women in Uganda.
Between September 2002 and November 2004, we enrolled 1275 young women attending the Naguru Teenage Information and Health Centre, located in a suburb of Kampala, Uganda, as part of a large HPV survey. Study methods are described elsewhere (Banura et al., 2008). Study participants were informed of the study aims and procedures and signed an informed consent form or assent form in the case of minors. The Institutional Review Boards of the Faculty of Medicine of Makerere University, the Uganda National Council of Science and Technology, and the International Agency for Research on Cancer approved the study.
The present report includes 111 women randomly selected among study participants. The distribution of the women included in the present report by year of recruitment, age (mean age 20 years; range 16–24), lifetime number of sexual partners (75 % reporting 
two partners) and HIV infection (eight women were HIV-positive) was well representative of the total study population.
From each woman, a sample was taken from the cervix by rotating (36 °) a sterile cotton swab thrice in the cervix, and smearing it within a 0.5–1.0 cm diameter on 3MM Whatman filter paper cut to the size of a small glass slide (5x2 cm). The cervical material remaining on the cotton swab was then placed into a 15 ml holding tube containing 5 ml PBS (pH 7.2). Paper samples were dried, placed in auto-seal (ziplock) plastic bags and stored at room temperature (25–30 °C), whereas PBS samples were kept at –20 °C until shipment to DDL Diagnostic Laboratory, Voorburg, the Netherlands, for DNA extraction and HPV genotyping.
Part of the filter paper was punched (
4 mm circle) and transferred to a 0.5 ml microcentrifuge tube containing 50 µl distilled water. DNA was released by boiling for 5 min. For PBS samples, DNA was isolated from 200 µl cell suspension by the MagNA Pure LC instrument (Roche Diagnostics), using the Total DNA isolation kit (Roche Diagnostics). DNA was eluted in 100 µl water, and 10 µl was used for each PCR reaction.
Every PCR reaction included positive and negative controls. Strict laboratory precautions and quality assurance/quality control measures were followed to avoid cross-contamination and carry-over PCR assay. The short PCR fragment (SPF)10 PCR primer set was used to amplify a broad spectrum of HPV genotypes, as described previously (Kleter et al., 1998, 1999). The same SPF10 amplimers were used to identify the HPV type by reverse hybridization on a reverse hybridization line probe assay (LiPA), containing probes for 25 different HPV types, including HPV6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70 and 74 (SPF10 HPV LiPA version 1; Labo Bio-medical Products).
To compare the types of samples, we calculated the kappa statistic for the detection of overall HPV positivity and presence of each individual HPV type and of multiple-type infections in paper and PBS samples from the same woman.
Overall, the prevalence of any HPV type was 82.9 % in PBS compared to 32.4 % in paper samples (kappa statistic=0.18) (Table 1
). Twenty-two different HPV types were detected in 225 infections in PBS samples, but only 18 types were found in 68 infections in paper samples. The most frequently detected high-risk HPV types were HPV52 (22.5 % in PBS, 9 % in paper and 7.2 % in both samples), HPV18 (18.9 % in PBS, 2.7 % in paper and 0.9 % in both samples) and HPV16 (16.2 % in PBS, 4.5 % in paper and 2.7 % in both samples). The most frequently detected low-risk type was HPV6 (20.7 % in PBS, 9 % in paper and 7.2 % in both samples). The majority of kappa statistics for the detection of individual HPV types fell between 0.20 and 0.40, and hence suggested relatively poor agreement. Multiple-type infections were detected in 54.1 % of PBS samples compared to 15.3 % of paper samples. Infection with four HPV types was also relatively common (18.0 %) in PBS samples, but not in paper samples (2.7 %) (Table 1).
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Reasons for the disagreement between our findings and those of Kailash et al. (2002), which were much more favourable to paper samples, are unclear, but they could include differences in the load of cells and HPV DNA obtained in the two studies. All women in the Indian study had cervical dysplasia or carcinoma, whereas we included young women who were free of cervical lesions at visual inspection with acetic acid and, on account of their age, were unlikely to harbour cervical carcinoma. In addition, Kailash et al. (2002) tested for HPV16 only, whereas we tested for 25 HPV types, using a highly sensitive and well-validated PCR-based assay (Kleter et al., 1999). Conversely, DNA extraction was performed in a similar way in our study and in that of Kailash et al. (2002). Smearing the swab on paper, however, is a highly variable step in the procedure, and it is difficult to control spot size. Therefore, it remains unknown how many cells are placed on the paper, and how representative this paper sample is of the entire cervical swab sample.
Although it is conceivable that DNA extraction from paper samples can be improved, our results do not encourage, for the moment, the use of paper samples for HPV detection. To permit the use of paper samples, a standardized procedure should be developed to ensure equivalence with liquid-based samples.
ACKNOWLEDGEMENTS
Financial support was received from SIDA/SAREC (Sweden) for the collaboration between Makerere University and Karolinska Institutet, Stockholm, Sweden; the Naguru Teenage Information and Health Centre, Kampala, Uganda; ICRETT fellowship number ICR R 06/139; the Cancer Registry of Norway, Oslo, Norway; the International Agency for Research on Cancer, Lyon, France; and the Bill & Melinda Gates Foundation (grant number 35537).
REFERENCES
Banura, C., Franceschi, S., van Doorn, L. J., Arslan, A., Wabwire-Mangen, F., Mbidde, E. K., Quint, W. & Weiderpass, E. (2008). Infection with human papillomavirus and HIV among young women in Kampala, Uganda. J Infect Dis in press
Beebe, J. L. & Briggs, L. C. (1990). Evaluation of enzyme-linked immunoassay systems for detection of human immunodeficiency virus type 1 antibody from filter paper disks impregnated with whole blood. J Clin Microbiol 28, 808–810.
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Garrick, M. D., Dembure, P. & Guthrie, R. (1973). Sickle-cell anemia and other hemoglobinopathies: procedures and strategy for screening employing spots of blood on filter paper as specimens. N Engl J Med 288, 1265–1268.[Medline]
Gupta, B. P., Jayasuryan, N. & Jameel, S. (1992). Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification. J Clin Microbiol 30, 1913–1916.
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Kleter, B., van Doorn, L. J., ter Schegget, J., Schrauwen, L., van Krimpen, K., Burger, M., ter Harmsel, B. & Quint, W. (1998). Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses. Am J Pathol 153, 1731–1739.
Kleter, B., van Doorn, L. J., Schrauwen, L., Molijn, A., Sastrowijoto, S., ter Schegget, J., Lindeman, J., ter Harmsel, B., Burger, M. & Quint, W. (1999). Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus. J Clin Microbiol 37, 2508–2517.
Lorincz, A. T. (2006). HPV testing by hybrid capture. In Emerging Issues of HPV Infections: From Science to Practice, pp. 54–62. Edited by J. Monsonego. Basel: Karger.
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