Prevalence and type spectrum of human papillomaviruses in healthy skin samples collected in three continents

Annika Antonsson1, Cornelia Erfurt1, Kristina Hazard1, Veroniqa Holmgren1, Melinda Simon1, Akio Kataoka2, Shahadat Hossain3, Camilla Håkangård1 and Bengt Göran Hansson1

1 Department of Medical Microbiology, Malmö University Hospital, Lund University, SE-20502 Malmö, Sweden
2 3-3-35 Yatsushiro-Shi, Kumamoto 830, Japan
3 Department of Oncology, Bangabandhu Sheikh Mujib Medical University, Shahbagh, Dhaka, Bangladesh

Correspondence
Bengt Göran Hansson
bengt-goran.hansson{at}mikrobiol.mas.lu.se


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In order to investigate whether previous findings of ubiquitous skin papillomavirus infection in Caucasians apply to populations from other parts of the world, skin swab samples from Bangladesh, Japan, Ethiopia and Zambia were analysed in parallel with Swedish samples. The prevalence of HPV DNA in the material from Bangladesh was 68 %, Japan 54 %, Ethiopia 52 %, Zambia 42 % and Sweden 70 %. A great multiplicity of genotypes was demonstrated by the finding of 88 HPV types or putative types in 142 HPV DNA-positive samples in total. Double or multiple genotypes were frequently found in the same sample. The most prevalent HPV type was HPV-5, with an overall prevalence of 6·5 %. This was also the only type that was found in samples from all of the countries in the study. The results presented show that commensal skin HPV infections have a worldwide distribution with a very broad spectrum of genotypes.

The GenBank accession numbers of new sequences reported in this paper are AY009879, AY009880, AY009883, AY009884, AY009887, AY040275AY040280, AY040282AY040284, AF440445AF440450 and AF542099AF542103.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human papillomaviruses (HPVs) are small epitheliotropic DNA viruses that can induce cutaneous and mucosal lesions, and certain high-risk HPV types are the causative agents of anogenital cancers (zur Hausen, 1996). Epidermodysplasia verruciformis (EV) is a rare hereditary skin disease manifesting as multiple warts. A broad variety of HPV types, which have been referred to as EV HPVs, e.g. HPV-5, -8, -9, -12, -14, -15, -17 and -19–25 (Kremsdorf et al., 1984; Orth et al., 1978), has been found in these patients. Some skin HPV types, mainly HPV-5 and HPV-8, have been found in skin cancer lesions, for example in EV patients (Jablonska & Majewski, 1994, Orth et al., 1979) and in immunosuppressed patients (Bens et al., 1998; Berkhout et al., 2000; de Villiers et al., 1997), and have therefore been suggested to be high-risk HPVs for these patients. However, in recent years it has been found that skin HPV types are widely spread among humans, giving rise to subclinical infections generally without causing warts or other lesions (Antonsson et al., 2000; Astori et al., 1998; Boxman et al., 1997).

In previous reports, we have shown that normal skin of healthy Swedish adults and children is infected with a great number of different HPV types and putative types, and that colonization of the skin with HPV starts very early in life (Antonsson et al., 2000, 2003). In the present study, we have collected and analysed skin samples from healthy individuals from three continents in order to investigate whether there are any major differences in skin HPV prevalence or genotype distribution between temperate, subtropical and tropical geographical areas or, alternatively, between Caucasian and non-Caucasian population groups.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Subjects.
Specimens were taken from each of the following areas of five different countries: from healthy skin of 50 individuals from the cities of Dhaka, Bangladesh; Malmö, Sweden; Addis Abeba, Ethiopia; from a rural area in Zambia; and from 48 persons in the city of Kumamoto, Japan. Half of the samples were collected from women and half from men in each country. The age ranges of the individuals were 16–70 years (median 35) for Bangladesh; 15–77 years (median 43·5) for Japan; 16–71 years (median 39) for Sweden; and 18–53 years (median 32) for Zambia and 20–58 years (median 28·5) for Ethiopia. All samples were collected from healthy individuals who, on the whole, were assumed to be representative of the general population of the respective countries.

Samples.
Skin swab samples were collected with pre-wetted (0·9 % NaCl solution) cotton-tipped swabs (Bio Hospital, Kopparberg, Sweden), which were drawn back and forth five times over the forehead skin within an area of 5x10 cm and then suspended in 1 ml 0·9 % NaCl solution. The samples from Sweden were kept at 4 °C for a maximum of 24 h before being analysed, while the specimens from Bangladesh, Japan, Zambia and Ethiopia were kept at room temperature for a maximum of 7 days until they reached the laboratory and were analysed.

PCR and HPV type determination.
A PCR test with the primer pair FAP59/FAP64 was used for detection of skin HPV DNA, as previously described (Forslund et al., 1999). The protocol was followed as described except for the MgCl2 concentration, which was modified to 3·5 mM. The PCR products were cloned into the pCR-script SK(+) cloning vector (Stratagene). Between two and four clones per sample were sequenced with both forward and reverse primers (BigDye; Applied Biosystems) and the sequences obtained were compared with available sequences in the GenBank database using the BLAST server (http://www.ncbi.nlm.nih.gov/blast/).

The PCR products obtained spanned the HPV L1 gene from nt 6044 to 6480 (numbering relative to HPV 20). Since full-length L1 sequences were not obtained, the new HPV isolates detected in this study were designated putative HPV types. The guidelines from the Papillomavirus Nomenclature Committee 1995 (14th International Papillomavirus Conference, Quebec City, Quebec, Canada) were followed in defining a new putative HPV type (de Villiers, 2001).

All samples were also analysed for human DNA by PCR, targeting the L1 repetitive sequence (Deragon et al., 1990).

Statístical analysis.
The chi-square test was used to compare the prevalence of HPV DNA and human DNA among the different countries.

Nucleotide sequence accession numbers.
Sequences of FA46, FA48–49, FA52, FA56–60, FA62–64, FA73–78 and FA93–97 have been submitted to GenBank with the following accession numbers; FA46, AY009880; FA48, AY009879; FA49, AY009884; FA52, AY009883; FA56, AY040275; FA57, AY040276; FA58, AY040277; FA59, AY040278; FA60·1, AY040279; FA60·2, AY040280; FA62, AY040282; FA63, AY040283; FA64, AY040284; FA73, AF440445; FA74, AF440446; FA75, AF440447; FA76, AF44048; FA78, AF440450; FA93, AF542099; FA94, AF542100; FA95, AF542101; FA96, AF542102; FA97, AF542103.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bangladesh
Thirty-four (68 %), 17 men and 17 women, of the 50 individuals from Bangladesh were HPV DNA positive. Thirteen of the samples tested negative for human DNA, out of which 10 were HPV DNA positive. The HPV type determination revealed four new putative types (FA46, FA48, FA49 and FA52) together with 29 HPV types or previously reported putative types (Table 1). One sample from a male contained a subtype of FA20 (92 % sequence homology). It was not possible to determine the HPV type of two samples collected from males, despite several attempts.


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Table 1. HPV types and putative types found in samples collected in Bangladesh, Japan, Sweden, Zambia and Ethiopia

The total number of individuals with each specific HPV type is given together with human L1 DNA-positive and human L1 DNA-negative results (respectively) in parentheses.

 
Japan
HPV DNA was detected in 26 (54 %) of the 48 Japanese samples, collected from 14 men and 12 women. Thirty-one of the samples tested negative for human DNA and 20 of these were HPV DNA positive. The HPV DNA-positive specimens were HPV type determined and 20 previously described HPV types or putative types plus eight new putative HPV types were found (Table 1).

Sweden
Thirty-five (70 %) of the 50 Swedish samples were HPV DNA-positive, 20 from men and 15 from women. Five of the samples tested negative for human DNA and two of these were HPV DNA positive. HPV type determination of the positive specimens revealed 34 previously described HPV types or putative types and five new putative types, FA73–76 and FA78 (Table 1).

Zambia
Twenty-one (42 %), 14 men and 7 women, of the 50 Zambian individuals were HPV DNA positive. Twenty-nine of the specimens tested negative for human DNA by PCR, out of which 16 were HPV DNA positive. HPV type determination revealed two new putative types (FA93 and FA94) together with 11 previously described skin HPV types or putative types (Table 1). Two subtypes, one of FA27 (92 % sequence homology) and another of HPV-20 (91 % sequence homology) were found in samples from two males.

Ethiopia
Twenty-six (52 %) of the 50 Ethiopian samples were HPV DNA positive, 11 from men and 15 from women. Thirty-one of the samples tested negative for human DNA and 17 of these were HPV DNA positive. The HPV types were determined in the HPV DNA-positive specimens and 25 previously described HPV types or putative types plus three new putative HPV types (FA95–FA97) were found (Table 1). Two putative subtypes were detected, one of HPV-37 (92 % sequence homology) and another of FA25 (92 % sequence homology). Three samples could not be HPV type determined; one was collected from a male and two from females.

Summary
The HPV DNA prevalence found in the Zambian samples was significantly lower than that of the samples collected in Sweden (P<0·01) and in Bangladesh (P<0·05).

All of the putative HPV types that have not been reported previously, together with the types most closely related to them, are presented in Table 2.


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Table 2. Twenty-two new putative HPV types and their most closely related HPV type or putative type

 
Altogether, 142 samples proved positive for HPV DNA and 88 different HPV types or putative types were found, 22 of which have not been described before. Thirty-three different HPV types or putative types were seen in the samples from Bangladesh, 28 in the Japanese samples, 40 in the Swedish, 13 in the Zambian and 28 in the Ethiopian samples. Fifty of the 88 HPV types or putative types detected in this study were found in only one country (Table 1). Only one HPV type, HPV-5, was detected in samples from all five countries and HPV-5 was found as the single type or together with one or more other HPV types or putative types in 16 of the 248 samples analysed (6·5 %). HPV-47 and FA25 were also common and were detected in samples from all countries except Zambia.

Fifty-three (39 %) of the 137 HPV type-determined samples contained two or more HPV types or putative types: eight samples from Bangladesh, 14 from Japan, 18 from Sweden, four from Zambia and nine from Ethiopia.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The present study shows that the previously reported high prevalence and type diversity of skin papillomavirus in healthy skin of Caucasians (Antonsson et al., 2000, 2003) also applies to people from other parts of the world.

The quality of the cellular human DNA as determined by PCR amplification of the human L1 repetitive DNA sequence varied significantly (P<0·001) between the samples collected from the different countries, ranging from five negative Swedish samples to 29 in Zambia and 31 in Ethiopia and Japan. The Zambian, Ethiopian and Japanese samples had been handled without a cold chain, which might have caused the observed decrease in the number of samples positive in the human DNA test. However, even in samples that were negative in the human DNA PCR test, we were able to detect HPV DNA at as high a prevalence as in the human DNA-positive samples, indicating that the viral DNA contained in the virions was protected against DNA degradation, contrary to the human cellular DNA in the samples.

Altogether, 88 different HPV types or putative types were found and of these 38 were detected in at least two countries. However, the majority of the HPV types and putative types were seen in one of the countries only. Furthermore, of the 137 HPV type-determined specimens, 53 (39 %) contained two or more HPV types or putative types each.

It is worth noting that five different subtypes of previously described HPV types or putative types were found in specimens from Bangladesh, Zambia and Ethiopia. In our previous studies spanning some 1000 HPV type determinations by DNA sequencing, we have only been confronted with putative HPV subtypes twice (Antonsson et al., 2000), which involved 90–98 % sequence homology with a previously described HPV type. However, the two putative types that were found previously had a sequence homology close to 98 %, while the sequence homology of the subtypes detected in this study was between 91 and 92 %.

The findings in this and previous studies together emphasize the ubiquity and impressive multiplicity of genotypes amongst the skin papillomaviruses, both in humans (Antonsson et al., 2000, 2003) and in animals (Antonsson & Hansson, 2002). Adapted to their hosts presumably over hundreds of millions of years, these viruses are the first viral skin commensals to be described.


   ACKNOWLEDGEMENTS
 
We thank Dominique Bengtsson, Haileyesus Behre and Abebe Tefera Kefale for help with obtaining the Ethiopian samples. This work was supported by the Cancer Foundation of Malmö University Hospital and the Alfred Österlund Foundation.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Antonsson, A. & Hansson, B. G. (2002). Healthy skin of many animal species harbors papillomaviruses which are closely related to their human counterparts. J Virol 76, 12537–12542.[Abstract/Free Full Text]

Antonsson, A., Forslund, O., Ekberg, H., Sterner, G. & Hansson, B. G. (2000). The ubiquity and impressive genomic diversity of human skin papillomaviruses suggest a commensalic nature of these viruses. J Virol 74, 11636–11641.[Abstract/Free Full Text]

Antonsson, A., Karanfilovska, S., Lindqvist, P. & Hansson, B.G. (2003). General acquisition of human skin papillomavirus infections occurs in early infancy. J Clin Microbiol (in press).

Astori, G., Lavergne, D., Benton, C., Hockmayr, B., Egawa, K., Garbe, C. & de Villiers, E. M. (1998). Human papillomaviruses are commonly found in normal skin of immunocompetent hosts. J Investig Dermatol 110, 752–755.[Abstract]

Bens, G., Wieland, U., Hofmann, A., Hopfl, R. & Pfister, H. (1998). Detection of new human papillomavirus sequences in skin lesions of a renal transplant recipient and characterization of one complete genome related to epidermodysplasia verruciformis-associated types. J Gen Virol 79, 779–787.[Abstract]

Berkhout, R. J., Bouwes Bavinck, J. N. & ter Schegget, J. (2000). Persistence of human papillomavirus DNA in benign and (pre)malignant skin lesions from renal transplant recipients. J Clin Microbiol 38, 2087–2096.[Abstract/Free Full Text]

Boxman, I. L., Berkhout, R. J., Mulder, L. H., Wolkers, M. C., Bouwes Bavinck, J. N., Vermeer, B. J. & ter Schegget, J. (1997). Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers. J Investig Dermatol 108, 712–715.[Abstract]

Deragon, J. M., Sinnett, D., Mitchell, G., Potier, M. & Labuda, D. (1990). Use of gamma irradiation to eliminate DNA contamination for PCR. Nucleic Acids Res 18, 6149.[Medline]

de Villiers, E. M. (2001). Taxonomic classification of papillomaviruses. Papilloma Rep 12, 57–63.

de Villiers, E. M., Lavergne, D., McLaren, K. & Benton, E. C. (1997). Prevailing papillomavirus types in non-melanoma carcinomas of the skin in renal allograft recipients. Int J Cancer 73, 356–361.[CrossRef][Medline]

Forslund, O., Antonsson, A., Nordin, P., Stenquist, B. & Hansson, B. G. (1999). A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin. J Gen Virol 80, 2437–2443.[Abstract/Free Full Text]

Jablonska, S. & Majewski, S. (1994). Epidermodysplasia verruciformis: immunological and clinical aspects. Curr Top Microbiol Immunol 186, 157–175.[Medline]

Kremsdorf, D., Favre, M., Jablonska, S., Obalek, S., Rueda, L. A., Lutzner, M. A., Blanchet-Bardon, C., Van Voorst Vader, P. C. & Orth, G. (1984). Molecular cloning and characterization of the genomes of nine newly recognized human papillomavirus types associated with epidermodysplasia verruciformis. J Virol 52, 1013–1018.[Medline]

Orth, G., Jablonska, S., Favre, M., Croissant, O., Jarzabek-Chorzelska, M. & Rzesa, G. (1978). Characterization of two types of human papillomaviruses in lesions of epidermodysplasia verruciformis. Proc Natl Acad Sci U S A 75, 1537–1541.[Abstract]

Orth, G., Jablonska, S., Jarzabek-Chorzelska, M., Obalek, S., Rzesa, G., Favre, M. & Croissant, O. (1979). Characteristics of the lesions and risk of malignant conversion associated with the type of human papillomavirus involved in epidermodysplasia verruciformis. Cancer Res 39, 1074–1082.[Medline]

zur Hausen, H. (1996). Papillomavirus infections – a major cause of human cancers. Biochim Biophys Acta 1288, F55–78.[CrossRef][Medline]

Received 18 September 2002; accepted 10 March 2003.