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Quantitative Analysis of KSHV (HHV-8) Virus Load in Plasma, PBMC and Lymph Nodes from HIV-1-Infected Persons T. B. CAMPBELL*, K. A. STASKUS, R. EVANS, I. E. WHITE, X. Q. ZHANG, J. FOLKVORD, and E. CONNICK.
Univ. of Colorado Hlth. Sci. Ctr., Denver and Univ. of Minnesota, Minneapolis Previous studies on the distribution of Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) in body compartments have been limited by the use of qualitative or semi-quantitative measures of KSHV virus load. We have used a real-time polymerase chain reaction assay (Taqman PCR) to quantify KSHV DNA in the peripheral blood and lymphoid tissue of HIV-1-infected persons. The assay provided reproducible quantitation of KSHV-infected cells for a range of infected cell frequencies from 0.1 to 10-5 and quantitation of KSHV DNA in plasma was reproducible between 100 to 106 copies/mL. The 95% confidence interval for the intersample variance of replicate cell and plasma samples was +0.2 log1010.
Real-time PCR was used to quantify KSHV DNA in paired plasma, PBMC and lymph nodes samples from 18 HIV-1-infected persons (16 male, 2 female; median age 33 y) who did not have Kaposi's sarcoma. Quantifiable levels of KSHV DNA were detected in zero of 18 plasma and in only 1 of 18 PBMC specimens from HIV-1 infected subjects. KSHV DNA was not detected and in paired plasma and PBMC samples from 8 HIV-1/KSHV-seronegative controls. In contrast, quantifiable KSHV DNA was detected in 7 of 18 lymph node specimens from HIV-1-infected subjects (median 31 copies/105 cells; range 10 to 100 copies/105 cells). KSHV gene expression was detected in cells in the lymph node with 100 copies/105 cells by in situ hybridization with a probe for the KSHV T0.7 RNA, but not with probes for ORF73, v-cyclin, T1.1 or v-IL6 RNAs. Among the 7 subjects with quantifiable levels of KSHV DNA in lymph nodes, only 2 subjects were KSHV LANA seropositive and KSHV lymph node DNA levels were not related to plasma HIV-1 RNA levels or CD4+ lymphocyte counts (P>0.5; Spearman's rank correlation). These findings suggest that KSHV-infected cells are commonly present in low frequencies in lymph tissues of HIV-1 infected persons who do not have KS. PCR analysis of lymphoid tissue may detect KSHV infection that is not apparent by measurement of LANA antibodies or PCR amplification of KSHV DNA in PBMC.
Key Words: HHV-8, KSHV, PCR
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