PCR with the Aqueous Humor, Blood Leukocytes and Vitreous of Patients Affected by Cytomegalovirus Retinitis and Immune Recovery Uveitis

Siqueira, Rubens Camargo; Cunha, Aldo Albuquerque; Oréfice, Fernando; Campos, Wesley Ribeiro; Figueiredo, Luíz Tadeu Moraes

doi:10.1159/000074566

Cited by 9 publications

(2 citation statements)

References 15 publications

(14 reference statements)

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“…A recent study on HIV-positive patients with IRU showed the absence of CMV genome on the PCR analysis, supporting the hypothesis that IRU is not directly related to viral replication in the eye (6).…”

Section: Discussionsupporting

confidence: 54%

Immune Recovery Uveitis in a iatrogenically Immunosuppressed Patient

Miserocchi

Modorati

Brancato

2005

European Journal of Ophthalmology

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Section: Discussionsupporting

confidence: 54%

Immune Recovery Uveitis in a iatrogenically Immunosuppressed Patient

Miserocchi

Modorati

Brancato

2005

European Journal of Ophthalmology

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“…We are indebted to Klaus Roemer for critical review of the manuscript. GAA ACG CGC G C GC AAT CGG 81873-81890 2, 3, 9, 12, 22 R2 (9,12,22) TAC GCT GCA GTT CAC/CCC AG 82055-82036 2 R3 (11) T-G AAC TGG AAC GTT TGG C 82174-82156 2, 8, 9, 12, 13, 22 F4 (18, 19) CGG AAA CGA TGG TGT AGT TGG 82571-82591 R5 (18,19) TCC AAC ACC CAC T/AG ACC GGT 82838-82818 F6 (15) ATA GGA GGC GCC ACG TAT TC …”

mentioning

confidence: 99%

Errors in Published Sequences of Human Cytomegalovirus Primers and Probes: Do We Need More Quality Control?

2005

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Over the past decade, many authors have focused on PCR as a powerful technique for the evaluation of human cytomegalovirus (CMV). The key to PCR lies in the design of oligonucleotides, as the specific sequences largely affect PCR's efficacy and sensitivity. This study was designed to examine the quality of published sequences of CMV primers and probes.PubMed was searched in the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) for English peer-reviewed articles using CMV and PCR as keywords. Articles reporting on virus genotyping or species-level identification, as well as letters to editors and reviews were excluded. The full texts of 91 papers published between 1993 and 2004 were studied. Of these, 34 papers did not describe the detailed nucleotide sequence, including 17 papers using commercially available kits. The remaining 57 papers with a total of 199 CMV-specific oligonucleotides were examined. Oligonucleotides with identical sequences or with one additional nucleotide at either the 3Ј or 5Ј end of the sequence were identified as synonymous.Using The Sequence Manipulation Suite web-based programs (written by Paul Stothard, University of Alberta, Canada), the binding sites of all 199 oligonucleotides were identified using GenBank strain AD169 genome sequence (GenBank accession no. X17403 and NC_001347). Mismatches to all published sequences of CMV were analyzed by the Basic Local Alignment Search Tool (BLAST) (http://www .ncbi.nlm.nih.gov/BLAST/). Papers containing oligonucleotide errors were studied for technical reasons for employment of mutated oligonucleotides or subsequently published errata. Moreover, information on identified errors was conveyed to all corresponding authors and coauthors.Ten oligonucleotides did not match any CMV strain, and reasons were specified neither in the respective articles nor in authors' replies (Table 1). Primers R2, F6, and F9 were incorrectly transferred from previous publications (2, 5, 7) as indicated by the authors (V. H. Aquino and X. L. Pang, personal communication). The oligonucleotides F1, R3, and P10 were apparently also incorrectly transferred from prior publications (14). Moreover, primers F4 and R5 possessed mismatches at their 3Ј-end triplets, which may reduce PCR efficiency (21). Furthermore, the two degenerated probes (P7 and P8) contained Y (C/T) instead of R (A/G) in P7 and vice versa in P8. Obviously, these mismatches may drastically affect the efficacies of these probes (17).In summary, we show that 5% of the CMV oligonucleotides included mismatches to all published sequences half of which were incorrectly reported mostly in secondary publications. Moreover, since we considered all sequences correct when they matched at least one CMV strain, even when the authors used a different strain for design or admitted an error, the number of errors is underestimated by our approach. These observations point to the possibility that the report of erroneous primers and probes is a widespread problem irritating other researchers. I...

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Cytomegalovirus antibodies in tear fluid of patients with retinitis

et al. 2006

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The diagnosis of cytomegalovirus retinitis (CMV-R) is difficult and usually based on clinical criteria or invasive diagnostic procedures. The purpose of this study was to investigate a possible association between CMV-R and specific anti-CMV antibodies in tears. Paired tear and serum samples were obtained from 96 individuals, which included 20 children with congenital CMV infection and chorioretinitis, 56 adults with retinitis with clinical signs suggestive of viral infection, and 20 healthy control subjects, and were tested for CMV antibodies using ELISA. The prevalence of anti-CMV antibodies in tears was found to be 80% (16/20) in children, 35% (20/56) in adults, and 5% (1/20) in control subjects. Furthermore, high antibody levels were detected in 35% (7/20) of children and 10.7% (6/56) of adults with retinitis, and were not found in control subjects. There was a strong association between high tear levels of anti-CMV antibodies and active ocular infection. No correlations were found between tear and serum antibodies. ELISA sensitivity was 80% and specificity 95%. Further studies are needed to compare the tear and intraocular levels of CMV-specific antibodies in patients with retinitis to find out if CMV antibody testing in tear fluid could substitute for more invasive diagnostic procedures.

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PCR with the Aqueous Humor, Blood Leukocytes and Vitreous of Patients Affected by Cytomegalovirus Retinitis and Immune Recovery Uveitis

Cited by 9 publications

References 15 publications

Immune Recovery Uveitis in a iatrogenically Immunosuppressed Patient

Immune Recovery Uveitis in a iatrogenically Immunosuppressed Patient

Errors in Published Sequences of Human Cytomegalovirus Primers and Probes: Do We Need More Quality Control?

Cytomegalovirus antibodies in tear fluid of patients with retinitis

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