IN-SITU DETECTION OF HUMAN CYTOMEGALOVIRUS DNA IN GASTROINTESTINAL BIOPSIES FROM AIDS PATIENTS BY MEANS OF VARIOUS PCR-DERIVED METHODS

The development of new in situ assessment of HCMV disease on endoscopi cal gastrointestinal biopsies from AIDS patients is described and comp ared with the viral load measured by semiquantitative solution-phase P CR (SQ-PCR). Ten biopsies were examined by viral isolation, standard h istology, in situ hybridization (ISH), in situ PCR-hybridization (PCR- ISH) and SQ-PCR, using the same target sequence. The methods developed for in situ HCMV detection were HCMV primers, the plasmid pCMV 406-S, a vector-free-digoxigenin-labelled HCMV-362 probe and the pSK + MCS n onsense probe. Paraffin-embedded MRCS cells, either HCMV-infected or u ninfected served as controls of specificity for ISH. beta-Actin primer s were designed as markers of DNA integrity. Computerized models of th e PCR, solution-phase and in situ PCR on formalin-fixed DNA indicated that HCMV and beta-actin primers were efficient and specific. Nine bio psies were negative for HCMV by histology and virus isolation. SQ-PCR revealed 80 000; 80 and <80 HCMV genomic equivalents in 6, 2 and 2 bio psies, respectively. In 8 biopsies, both ISH and PCR-ISH identified po sitive nuclei in the intestinal epithelium, with sparing of the lamina propria. This indicates that an improvement in in situ methods can he lp the timely diagnosis of HCMV infection. Direct in situ PCR with bet a-actin primers showed a positive signal in all the nuclei in the tiss ue sections, whereas omission of Tag polymerase resulted in an absence of signal, implying optimal in situ PCR. The data suggest an early-st age reactivation of HCMV, possibly harboured in the intestinal epithel ium.