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Sir, The possible role of human herpesvirus (HHV)-8 has monopolized the scienti¢c debate on the etiopathogenesis of Kaposi sarcoma (KS) in recent months (1). Studies on other HHVs, are very few and inconclusive.We analysed DNA in tissue samples from patients with histologically proven KS for the presence of HHV-6, HHV-7, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and HSV DNA sequences. The study involved 23 patients (49 to 84 years of age, 17 men and 6 women) with Mediterranean KS and 3 renal transplant male patients (56 to 64 years of age) who had developed KS after being treated with prednisone and cyclosporine. All were negative for HIV antibodies by ELISA and Western blot. The DNA was extracted from fresh tissues or from sections of formalin-¢xed para¤n-embedded skin specimens collected between 1983 and 1997. The samples were tested for the presence of HHV-6, HHV-7, EBV, CMV and HSV sequences by polymerase chain reaction (PCR) with speci¢c primers (2 ^ 4). DNAwas obtained from skin by proteinase K sodium-dodecyl-sulphate digestion followed by phenol-chloroform extraction and ethanol precipitation of nucleic acids. The HHV-6 sequences of the outer primer were 5'CCGCAATCGAATCCACCTAGCGG-3' and 5'-GTGAGAACGGATTCGAACAGTGCTG-3'. Inner primers were 5'-CCCATTTACGATTTCCTGCACCACCTCTCTGC-3' and 5'-TTCAGGGACCGTTATGTCATTGAGCATGTCG-3'. The thermocycling procedure (Thermal Cycler 9600 Perkin-Elmer Cetus, Norwalk, CT) consisted of initial denaturation at 943C for 2 min, 30 cycles of denaturation at 943C for 30 sec, annealing at 553C for 30 sec, extension at 723C for 30 sec and ¢nal extension at 723C for 2 min. The same cycles were used for the bath inner primer sets. HHV-6 positive control DNA was obtained by standard phenol extraction of HHV-6 strain GS-infected HSB-2 cells. Negative control DNA was extracted from uninfected HSB-2 cells. The HHV-7 sequences of the outer primer were 5'-AGTTCCAGCACTGCAATCG-3' and 5'-CACAAAAGCTCGCTATCAA-3'. The inner primers were 5'-CGCATACACCAACCCTACTG-3' and 5'-GACTCATTATGGGGATCGAC-3'. The HHV-7 DNA thermal condition included initial denaturation at 943C for 2 min, denaturation at 943C for 30 sec, annealing at 603C for 30 sec, extension at 723C for 30 sec for 30 cycles, and ¢nal extension at 723C for 2 min. The same cycles were used for the bath inner primer sets. HHV-7 positive control DNA was obtained by extraction of HHV-7 straininfected Sup-T1 cells. Negative control DNA was extracted from non-infected Sup-T1 cells. Nested PCR products of HHV-6 and HHV-7 were visualized by electrophoresis on 2% agarose gels (in TBE bu¡er and containing 1 mg of ethidium bromide). From sequence analyses, the sizes of the products after nested PCR were expected to be 186 pb for HHV-6 and 264 pb for HHV-7. PCR conditions for the detection of HSV, CMVand EBV were as previously described (2). Sequences of HHV-7, HHV-6, HSV, EBV and CMV DNA were not detected in patients' lesional skin. In KS, the ultimate evidence of a causal role for HHV-8 is still lacking. Among many aspects of HHV-8 biological properties still to be clari¢ed are its serological prevalence in the general population, in which of the host cells the virus may remain latent and those in which it may multiplicate, and the factors disturbing the virus ^ host balance. Other HHVs, therefore, may be important in KS. For example, CMV, EBV, HHV-6 and HHV-7 alone or in association may act as co-factors and transactivators. Only a handful of studies have veri¢ed such a possibility. CMV, HHV-6 and HHV-7 have been detected in skin specimens from AIDS-associated KS (5), though their prevalence was similar or lower than in normal skin of AIDS patients without KS. In 7 of 22 classic KS, EBV has been found in the lesional skin and in 4 of them it was associated with HHV-8 (6). Lastly, such an association has also been found in a single patient with iatrogenic KS (7). In our study, which is probably the largest one on nonAIDS-associated KS for HHVs other than HHV-8, we were unable to con¢rm that HSV, CMV, EBV, HHV-6 and HHV-7 play an aetiological role.

Non-AIDS-related Kaposi Sarcoma Tissues do not Contain DNA Sequences of HHV-6, HHV-7, Epstein-Barr Virus, Cytomegalovirus and HSV