Polymerase Chain Reaction Detection of Kaposi's Sarcoma-Associated Herpesvirus-Optimized Protocols and Their Application to Myeloma

Abstract

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 multicentric Castleman's disease has been well established, its presence in some other diseases, such as multiple myeloma, remains controversial. Because most KSHV studies are based on polymerase chain reaction (PCR) analysis, the conflicting data may be attributable to variations in the methods, primer sets, and target sequences selected. To establish an efficient and reliable PCR approach for KSHV detection we designed eight sets of primers to six regions (ORFK1, ORFK2, ORFK9, ORK26, ORF72, and ORF74) of the KSHV genome using 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 lowest 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 increase the detection efficiency, while nested PCR protocols are likely to introduce false positivity. Using ORFK9, ORF26 and ORF72 primer sets, we screened bone marrow biopsies from 18 cases of multiple myeloma, and failed to detect any KSHV sequences. This finding supports the conclusion that KSHV is not associated with multiple myeloma. Indeed, our results further confirm that although KSHV is universally present in Kaposi's sarcoma and primary effusion lymphoma, it is not ubiquitious.

Absence of human herpesvirus-8 in pulmonary inflammatory myofibroblastic tumor: immunohistochemical and molecular analysis of 20 cases

“…As we found no viral DNA, it was not surprising that all our cases were negative for latent nuclear antigen, corroborating the immunohistochemical findings on inflammatory myofibroblastic tumor from varying sites reported by Yamamoto et al 22 and pediatric cases reported by Mergan et al 23 Molecular identification of HHV-8 is known to produce both false-positive and false-negative results. [24][25][26] The latter is thought, in part, to be related to HHV-8 sequence variation, which can range up to 35% in certain regions of the viral genome, as in ORFK1. 26 The use of a single primer set based on a sequence with a high level of sequence variation can lower the PCR sensitivity and, therefore, the rate of HHV-8 detection.…”

“…24 False-positive results are thought to be due to utilization of high cycle protocols and/or complimentary primer sets, as in nested PCR. Through a set of validation tests, Pan et al, 25 demonstrated that nested PCR is readily contaminated, even in strictly controlled environments, and use of standard PCR with more than one set of primers is recommended. In their study, 6 of 16 negative controls tested positive for HHV-8 sequences in nested PCR using primers to ORF26, but none were positive utilizing standard PCR.…”

Kaposi's sarcoma-associated herpesvirus infection and Kaposi's sarcoma in Brazil

“…However, in a study by Pan and coworkers (28), the authors concluded that KSHV detection by PCR may involve a high rate of false-negative results. For this reason, optimization of PCR procedures is mandatory, and nested-PCR approaches as well as amplification of different segments of the KSHV genome are recommended in order to obtain accurate results (28). Using a standard PCR procedure to detect the KSHV genome in DNA from formalin-fixed paraffinembedded tissue from few cases, Caterinode-Araujo et al (25) initially found the virus in 3/7 (42.9%) KS cases; however, all of their samples were found to be positive after nested-PCR.…”

“…The samples without amplification in this first PCR assay were used in ORF26-directed nested-PCR (28). Doubtful cases were analyzed for amplification of viral ORF72 segment, which encodes a viral capsid protein (28).…”

“…The samples without amplification in this first PCR assay were used in ORF26-directed nested-PCR (28). Doubtful cases were analyzed for amplification of viral ORF72 segment, which encodes a viral capsid protein (28). Samples negative for all PCR assays were further evaluated for amplification of the ß-chain human globin gene with PCO 3 +/ PCO 4 + primers (29).…”