In Malawian patients with Kaposi sarcoma (KS) and their relatives, we investigated nucleotide-sequence variation in human herpesvirus-8 (HHV-8) subgenomic DNA, amplified from oral and blood samples by use of polymerase chain reaction. Twenty-four people had amplifiable HHV-8 DNA in >1 sample; 9 (38%) were seropositive for human immunodeficiency virus type 1, 21 (88%) were anti-HHV-8-seropositive, and 7 (29%) had KS. Sequence variation was sought in 3 loci of the HHV-8 genome: the internal repeat domain of open-reading frame (ORF) 73, the KS330 segment of ORF 26, and variable region 1 of ORF K1. Significant intraperson/intersample and intrasample sequence polymorphisms were observed in 14 people (60%). For 3 patients with KS, intraperson genotypic differences, arising from nucleotide sequence variations in ORFs 26 and K1, were found in blood and oral samples. For 2 other patients with KS and for 9 people without KS, intraperson genotypic and subgenotypic differences, originating predominantly from ORF K1, were found in oral samples; for the 2 patients with KS and for 4 individuals without KS, intrasample carriage of distinct ORF K1 sequences also were discernible. Our findings imply HHV-8 superinfection.

Human herpesvirus 8 (HHV-8Infection by human herpesvirus 8 (HHV-8) appears restricted to selected populations in the West and around the Mediterranean but is widespread in many sub-Saharan African countries (6). We studied HHV-8 subgenomic sequences amplified from oral and blood samples of Malawian patients with Kaposi's sarcoma (KS) and their family members and reported the inferences we made on how HHV-8 might be transmitted in a setting where HHV-8 is hyperendemic (5). Urine is another body fluid into which HHV-8 is shed (3). To determine the extent to which urine contributes to HHV-8 transmission, we evaluated intraindividual HHV-8 subgenomic differences between the urine and oral fluid.Urine samples were taken from the Malawian study group from a previous report (5). Nucleic acid in the cellular fraction was extracted using a QIAamp kit (QIAGEN, Santa Clarita, Calif.). Nested PCR (5) was then applied to the extract to amplify a 246-bp DNA segment in open reading frame K1. This segment encompasses the highly variable V1 region (spanning positions 366 to 577) (8) and is here referred to as K1/V1. Of 78 people from whom urine samples could be tested, K1/V1 DNA was amplifiable in 2 of 20 (10%) patients with KS and 3 of 58 family members (5.2%) (overall rate, 6.4%).The five individuals who carried urinary K1/V1 DNA were identified as B5 (male, 7 years of age), G2 (male, 23 years), I1 (male, 12 years), Ni (female, 30 years), and Ui (female, 32 years), following the identification system described in the previous publication (5) (index cases of KS are assigned the "i" suffix, and their family members are assigned numerals; letters denote family assignments). B5, G2, and I1 were seronegative, while Ni and Ui were seropositive for human immunodeficiency virus 1. We proceeded to assess the spectrum of K1/V1 sequences in the urine of these individuals. As B5 and G2 had previously been identified to carry amplifiable K1/V1 DNA in the mouth rinse (5), we included their rinse samples into the evaluation.DNA extracts from the urine and rinse samples were subjected to nested PCR using an EXPAND high-fidelity PCR system (Roche Diagnostics, Indianapolis, Ind.) (2). Clones were then generated from each K1/V1 PCR product using a TOPO TA cloning system (Invitrogen, Carlsbad, Calif.). From each colony, another round of PCR was carried out under conditions identical to the second-round high-fidelity PCR, except that a GC-rich clamping primer was used in place of the inner sense primer. The PCR products were subjected to denaturing gradient gel electrophoresis (DGGE) followed by nucleotide sequencing carried out as described previously (2). Figure 1 is a composite of representative DGGE gel pictures showing migration distances attained by the K1/V1 amplicons (DNA bands were revealed following SYBR Green I staining and UV transillumination). All PCR products yielding minority banding positions and some products representing the majority were sequenced (Fig. 1). The extent of K1/V1 nucleotide sequence diversity is depicted in Fig. 2...

Sequence polymorphisms in the gN and gO genes of cytomegalovirus (CMV) amplified from mouth rinse and urine samples of 19 Malawian patients with Kaposi's sarcoma (KS) and 58 of their first-degree relatives were investigated. CMV-DNA was amplified from 41 people (53%) from either the gN or gO region in at least one sample, from 14 people (18%) in both domains in at least one sample, and from 13 (17%) in either domain in both samples. Twenty-one (51%) were seropositive for human immunodeficiency virus-1 (HIV). Identical gN sequences were recovered from eight families and non-identical sequences in six, while identical gO sequences were found in three families and non-identical sequences in five. Five people, four of whom were children, each carried multitypic gN sequences or gO sequences. The findings are consistent with CMV spread along intra- and extra-household routes, and with multiple intra-host CMV infection.