734
well documented.1,2 However, this phase of the disease is often overlooked. Skin biopsy may show minimum infiltration, as in our case. Furthermore, the proportion of abnormal lymphocytes in the peripheral blood may be as low as 0.5%.1 Molecular analysis by PCR is a useful adjunct in the detection of HTLV-1 infection, especially when only small samples are available, or where serological studies are negative.3 It has also proved useful to distinguish dual infections with type C retroviruses.4 In the subclinical stage of the disease viral integration is usually polyclonal, whereas in the smouldering and acute phases of ATLL it is monoclonal.The two patterns can be distinguished only by Southern blot analysis, and not by PCR.’ This case and that of Bunker et al demonstrate the importance of HTLV-1 antibody status and careful scrutiny of skin biopsy specimens in patients with the requisite ethnic and geographical background who present with ill-defined papular, nodular, or erythematous skin lesions. Close inspection of the peripheral blood smear may help in the diagnosis of the subclinical state of the population at risk. Departments of Haematological Medicine, Dermatology, and Histopathology, King’s College Hospital, London SE5 9PJ, UK
1.
A. PAGLIUCA H. WILLIAMS J. SALISBURY G. J. MUFTI
Yamaguchi K, Nishimura H, Kohrogi H, Jono M, Miyamoto Y, Takatsuki K. A proposal for smoldering adult T-cell leukemia: a clinicopathologic study of five
cases. Blood 1983; 62: 758-66. 2. Kawano F, Yamaguchi K, Nishimura H, Tsuda H, Takatsuki K. Variation in the clinical courses of adult T-cell leukemia. Cancer 1985; 55: 851-56. 3. Kuefler PR, Bunn PA. Adult T cell leukaemia/lymphoma. Clin Haematol 1986; 15: 695-727. 4. Yamaguchi K, Kiyowaka T, Nakada K, et al. Polyclonal integration of HTLV-1 proviral DNA in lymphocytes from HTLV-1 seropositive individuals: an intermediate state between the healthy carrier state and smouldering ATL. Br J Haematol 1988; 68: 169-74. 5. Kumar De B, Srinivasan A. Detection of human immunodeficiency virus (HIV) and human lymphotropic virus (HTLV) type I or II dual infections by polymerase chain reaction Oncogene 1989; 4: 1533-35.
Looking for human papillomavirus type 16 by PCR SIR,-Several groups, using polymerase chain reaction (PCR) amplification methods, have reported very high rates of cervical infection with human papillomavirus type 16 (HPV-16).1-3 A variant ofHPV-16 ("16b") was reported to be common in the normal population2 but this finding has been retracted.4 We have used a PCR method based on consensus primers and the reported method for identifying HPV-16b, to investigate HPV-16 prevalence in several groups of women. Cervical swab samples were rapidly prepared for PCR by proteinase digestion. Volumes representing one-tenth and onefiftieth of the sample were analysed by the consensus primer amplification system6 This system provides amplification of a 450 bp region of the Ll open reading frame from a broad spectrum of HPV types. Oligonucleotide probes were used to distinguish HPV
amplification products from HPV-16-positive samples (9 samples from group A). Size marker lane M contains a mixture of PCR products from prototype HPV-16 and from "16b" (clone kindly provided by Dr P. Farrell). PCR products from clinical material, which includes cellular debris, detergent, and so on, typically migrate slightly slower than marker PCR products, which are derived from purified DNA. URR
types. The PCR and hybridisation analyses were done as described6 except that the consensus probe consisted of a mixture of 32P-Iabelled Ll PCR products (lacking MY11and MY09 primer regions) of HPV-16, HPV-18, and two unidentified genital HPVs. The sensitivity in the consensus primer analyses allowed the detection of as few as 100 copies of HPV per swab. In addition, amounts representing one-twentieth of each sample were amplified with HPV-16-specific primers for the identification of 16b (primers J and P2). The PCR products were analysed using acrylamide gel electrophoresis and ethidium bromide staining. The HPV-16 primers (specific for the upstream regulatory region [URR]) afforded the detection of as few as 500 copies of HPV-16 per swab. The results, for three groups of women, are summarised in the table. The prevalence of cervical HPV ranged from 31% to 44% (consensus primer PCR) and the prevalence of HPV-16 ranged from zero to 22% (either system, with perfect correlation). In every example of HPV-16, the URR amplification product was that of the prototype HPV-16 (examples are shown in the figure). We have analysed cervical swab samples from over 200 women and have found no evidence of HPV "16b". The prevalence of cervical HPV-16 infection which we found is much lower than that reported by others using PCR methods and the explanation probably lies in various forms of accidental contamination (or carryover) to which PCR is vulnerable. Rigorous procedures are essential and guidelines on how to avoid the misleading complications of carryover are available. 7,8
Cetus
Department of Infectious Diseases, Corporation, Emeryville, California, USA
MICHELE MANOS KENNETH LEE CATHERINE GREER
Lafayette, California
JEFFREY WALDMAN
Department of Medicine, University of Washington, Seattle
NANCY KIVIAT KING HOLMES
Department of Cell Biology, and New Mexico Tumor Registry, University of New Mexico, Albuquerque
COSETTE WHEELER
1. Bevan
HPV DETECTION RESULTS
*Group A=consecutive
women attending University of New Mexico women’s health clinic for routine annual gynaecological examinations. Group B = consecutIve women attending Planned Parenthood in Richmond, Califorma, for routine care not related to sexually transmitted disease, women with a history of genital warts or cervical dysplasia were excluded. Group C=consecutive women presenting to University of Washingtion student health clinic for routine gynaecological care
IS, Blomfield PI, Johnson MA, et al. Oncogenic viruses and cervical cancer. Lancet 1989; i: 907-08. 2. Tidy JA, Vousden KH, Farrell PJ. Relation between infection with a subtype of HPV16 and cervical neoplasia. Lancet 1989; i: 1225-27. 3. Ward P, Parry GN, Yule R, et al. Human papillomavirus subtype 16a. Lancet 1989; ii 170. 4. Tidy J, Farrell PJ. Retraction: human papillomavirus subtype 16b. Lancet 1989; u: 1535. 5. Manos MM, Ting Y, Wright DK, Lewis AJ, Broker TR, Wolinsky SM. The use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. In: Molecular diagnostics in human cancer. Cancer Cells 1989, 7: 209-14. 6. Ting Y, Manos M. Detection and typing of genital human papillomaviruses. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego: Academic Press, 1990: 356-67. 7. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-38 8. Kwok S. Procedures to minimize PCR-product carry-over. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego: Academic Press, 1990: 142-45.
well documented.1,2 However, this phase of the disease is often overlooked. Skin biopsy may show minimum infiltration, as in our case. Furthermore, the proportion of abnormal lymphocytes in the peripheral blood may be as low as 0.5%.1 Molecular analysis by PCR is a useful adjunct in the detection of HTLV-1 infection, especially when only small samples are available, or where serological studies are negative.3 It has also proved useful to distinguish dual infections with type C retroviruses.4 In the subclinical stage of the disease viral integration is usually polyclonal, whereas in the smouldering and acute phases of ATLL it is monoclonal.The two patterns can be distinguished only by Southern blot analysis, and not by PCR.’ This case and that of Bunker et al demonstrate the importance of HTLV-1 antibody status and careful scrutiny of skin biopsy specimens in patients with the requisite ethnic and geographical background who present with ill-defined papular, nodular, or erythematous skin lesions. Close inspection of the peripheral blood smear may help in the diagnosis of the subclinical state of the population at risk. Departments of Haematological Medicine, Dermatology, and Histopathology, King’s College Hospital, London SE5 9PJ, UK
1.
A. PAGLIUCA H. WILLIAMS J. SALISBURY G. J. MUFTI
Yamaguchi K, Nishimura H, Kohrogi H, Jono M, Miyamoto Y, Takatsuki K. A proposal for smoldering adult T-cell leukemia: a clinicopathologic study of five
cases. Blood 1983; 62: 758-66. 2. Kawano F, Yamaguchi K, Nishimura H, Tsuda H, Takatsuki K. Variation in the clinical courses of adult T-cell leukemia. Cancer 1985; 55: 851-56. 3. Kuefler PR, Bunn PA. Adult T cell leukaemia/lymphoma. Clin Haematol 1986; 15: 695-727. 4. Yamaguchi K, Kiyowaka T, Nakada K, et al. Polyclonal integration of HTLV-1 proviral DNA in lymphocytes from HTLV-1 seropositive individuals: an intermediate state between the healthy carrier state and smouldering ATL. Br J Haematol 1988; 68: 169-74. 5. Kumar De B, Srinivasan A. Detection of human immunodeficiency virus (HIV) and human lymphotropic virus (HTLV) type I or II dual infections by polymerase chain reaction Oncogene 1989; 4: 1533-35.
Looking for human papillomavirus type 16 by PCR SIR,-Several groups, using polymerase chain reaction (PCR) amplification methods, have reported very high rates of cervical infection with human papillomavirus type 16 (HPV-16).1-3 A variant ofHPV-16 ("16b") was reported to be common in the normal population2 but this finding has been retracted.4 We have used a PCR method based on consensus primers and the reported method for identifying HPV-16b, to investigate HPV-16 prevalence in several groups of women. Cervical swab samples were rapidly prepared for PCR by proteinase digestion. Volumes representing one-tenth and onefiftieth of the sample were analysed by the consensus primer amplification system6 This system provides amplification of a 450 bp region of the Ll open reading frame from a broad spectrum of HPV types. Oligonucleotide probes were used to distinguish HPV
amplification products from HPV-16-positive samples (9 samples from group A). Size marker lane M contains a mixture of PCR products from prototype HPV-16 and from "16b" (clone kindly provided by Dr P. Farrell). PCR products from clinical material, which includes cellular debris, detergent, and so on, typically migrate slightly slower than marker PCR products, which are derived from purified DNA. URR
types. The PCR and hybridisation analyses were done as described6 except that the consensus probe consisted of a mixture of 32P-Iabelled Ll PCR products (lacking MY11and MY09 primer regions) of HPV-16, HPV-18, and two unidentified genital HPVs. The sensitivity in the consensus primer analyses allowed the detection of as few as 100 copies of HPV per swab. In addition, amounts representing one-twentieth of each sample were amplified with HPV-16-specific primers for the identification of 16b (primers J and P2). The PCR products were analysed using acrylamide gel electrophoresis and ethidium bromide staining. The HPV-16 primers (specific for the upstream regulatory region [URR]) afforded the detection of as few as 500 copies of HPV-16 per swab. The results, for three groups of women, are summarised in the table. The prevalence of cervical HPV ranged from 31% to 44% (consensus primer PCR) and the prevalence of HPV-16 ranged from zero to 22% (either system, with perfect correlation). In every example of HPV-16, the URR amplification product was that of the prototype HPV-16 (examples are shown in the figure). We have analysed cervical swab samples from over 200 women and have found no evidence of HPV "16b". The prevalence of cervical HPV-16 infection which we found is much lower than that reported by others using PCR methods and the explanation probably lies in various forms of accidental contamination (or carryover) to which PCR is vulnerable. Rigorous procedures are essential and guidelines on how to avoid the misleading complications of carryover are available. 7,8
Cetus
Department of Infectious Diseases, Corporation, Emeryville, California, USA
MICHELE MANOS KENNETH LEE CATHERINE GREER
Lafayette, California
JEFFREY WALDMAN
Department of Medicine, University of Washington, Seattle
NANCY KIVIAT KING HOLMES
Department of Cell Biology, and New Mexico Tumor Registry, University of New Mexico, Albuquerque
COSETTE WHEELER
1. Bevan
HPV DETECTION RESULTS
*Group A=consecutive
women attending University of New Mexico women’s health clinic for routine annual gynaecological examinations. Group B = consecutIve women attending Planned Parenthood in Richmond, Califorma, for routine care not related to sexually transmitted disease, women with a history of genital warts or cervical dysplasia were excluded. Group C=consecutive women presenting to University of Washingtion student health clinic for routine gynaecological care
IS, Blomfield PI, Johnson MA, et al. Oncogenic viruses and cervical cancer. Lancet 1989; i: 907-08. 2. Tidy JA, Vousden KH, Farrell PJ. Relation between infection with a subtype of HPV16 and cervical neoplasia. Lancet 1989; i: 1225-27. 3. Ward P, Parry GN, Yule R, et al. Human papillomavirus subtype 16a. Lancet 1989; ii 170. 4. Tidy J, Farrell PJ. Retraction: human papillomavirus subtype 16b. Lancet 1989; u: 1535. 5. Manos MM, Ting Y, Wright DK, Lewis AJ, Broker TR, Wolinsky SM. The use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. In: Molecular diagnostics in human cancer. Cancer Cells 1989, 7: 209-14. 6. Ting Y, Manos M. Detection and typing of genital human papillomaviruses. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego: Academic Press, 1990: 356-67. 7. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-38 8. Kwok S. Procedures to minimize PCR-product carry-over. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols: a guide to methods and applications. San Diego: Academic Press, 1990: 142-45.
