RESUMO
BACKGROUND: Human cytomegalovirus (CMV) infection is a major cause of morbidity in transplant patients. Early diagnosis and treatment have been shown to improve outcome. We evaluated the suitability of CMV immediate early, early, and late gene expression detected by nucleic acid sequence-based amplification (NASBA) as markers of CMV infection. METHODS: Blood samples were taken immediately before transplant and every one to two weeks after transplantation for 12 weeks from 50 patients undergoing thoracic organ transplantation. CMV-NASBA was performed and results compared with serology, CMV pp65 antigenaemia (CMV-AG) and the development of clinical CMV infection. Patients received "preemptive" anti-CMV therapy with ganciclovir based on the CMV-AG results. RESULTS: CMV immediate early and early gene expression were detected in 87 and 47%, respectively, of patients without other evidence of CMV infection. CMV late gene expression had a sensitivity of 97% for infection (compared with 83% for CMV-AG P=0.06) and a specificity of 93% (compared with 100% P=NS). Late gene expression occurred at the same time as CMV antigenaemia but 1.1 weeks earlier than the threshold of antigenaemia (CMV-AG>10) used to initiate preemptive therapy. CONCLUSION: NASBA provided a standardized tool for the detection of CMV transcripts with a greater sensitivity than the standard antigenemia test. Detection of immediate early and early gene transcripts was not specific for subsequent infection. CMV late gene expression determined by NASBA was an accurate and early marker of CMV infection. Detection of CMV late gene expression could be used to trigger "preemptive" anti-CMV therapy.
Assuntos
Citomegalovirus/genética , Genes Virais/genética , Transplante de Coração , Transplante de Coração-Pulmão , Transplante de Pulmão , Replicação de Sequência Autossustentável/métodos , Adulto , Antivirais/uso terapêutico , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/imunologia , Feminino , Ganciclovir/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Transcrição GênicaRESUMO
Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.
Assuntos
Citomegalovirus/genética , Citomegalovirus/metabolismo , RNA Mensageiro/metabolismo , Replicação de Sequência Autossustentável/métodos , Proteínas Virais/metabolismo , Células Cultivadas/virologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Glicoproteínas , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Membrana , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/genéticaRESUMO
Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.