In situ detection of human cytomegalovirus DNA in gastrointestinal biopsies from AIDS patients by means of various PCR-derived methods.
J Virol Methods
; 56(2): 125-37, 1996 Feb.
Article
em En
| MEDLINE
| ID: mdl-8882643
ABSTRACT
The development of new in situ assessment of HCMV disease on endoscopical gastrointestinal biopsies from AIDS patients is described and compared with the viral load measured by semiquantitative solution-phase PCR (SQ-PCR). Ten biopsies were examined by viral isolation, standard histology, 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 nonsense probe. Paraffin-embedded MRC5 cells, either HCMV-infected or uninfected served as controls of specificity for ISH. beta-Actin primers were designed as markers of DNA integrity. Computerized models of the PCR, solution-phase and in situ PCR on formalin-fixed DNA indicated that HCMV and beta-actin primers were efficient and specific. Nine biopsies 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 biopsies, respectively. In 8 biopsies, both ISH and PCR-ISH identified positive nuclei in the intestinal epithelium, with sparing of the lamina propria. This indicates that an improvement in in situ methods can help the timely diagnosis of HCMV infection. Direct in situ PCR with beta-actin primers showed a positive signal in all the nuclei in the tissue sections, whereas omission of Taq polymerase resulted in an absence of signal, implying optimal in situ PCR. The data suggest an early-stage reactivation of HCMV, possibly harboured in the intestinal epithelium.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Viral
/
Reação em Cadeia da Polimerase
/
Infecções Oportunistas Relacionadas com a AIDS
/
Infecções por Citomegalovirus
/
Citomegalovirus
/
Sistema Digestório
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
J Virol Methods
Ano de publicação:
1996
Tipo de documento:
Article
País de afiliação:
França