Polymerase chain reaction detection of Kaposi's sarcoma-associated herpesvirus-optimized protocols and their application to myeloma

Since its discovery in 1994, KSHV (also called human herpesvirus-8 or HHV8) has been implicated in a variety of disorders. Although the association of KSHV with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and mult icentric Castleman's disease has been well established, its presence in som e other diseases, such as multiple myeloma, remains controversial. Because most KSHV studies are based on polymerase chain reaction (PCR) analysis, th e conflicting data may be attributable to variations in the methods, primer sets, and target sequences selected. To establish an efficient and reliabl e PCR approach for KSHV detection we designed eight sets of primers to six regions (ORFK1, ORFK2, ORFK9, OKK26, ORF72, and ORF74) of the KSHV genome u sing appropriate database and software. The detection sensitivity of these primers was carefully assessed and their reliability was strictly validated in a series of positive (15 KS and PEL samples) and negative (16 lymphoid tissues) controls. We found that primer sets to the ORFK9 region showed the highest sensitivity, whereas primer sets to ORFK1 and ORF74 showed the low est sensitivity. Primer sets to ORFK9, ORF26 and ORF72 regions detected all of the positive cases, whereas other primer sets showed varying detection rates or nonspecific bands. All 16 negative controls were negative with all primer sets. However, six of 16 negative controls became positive when we used nested PCR targeting ORF26,Therefore, multiple target KSHV sequences i ncrease the detection efficiency, while nested PCR protocols are likely to introduce false positivity. Using ORFK9, ORF26 and ORF72 primer sets, Re sc reened bone marrow biopsies from 18 cases of multiple myeloma, and failed t o detect any KSHV sequences. This finding supports the conclusion that KSHV is not associated with multiple myeloma. indeed, our results further confi rm that although KSHV is universally present in Kaposi's sarcoma and primar y effusion lymphoma, it is not ubiquitous.