Diagnosis of cytomegalovirus infections by qualitative and quantitative PCR in HIV infected patients

Cunha, Aldo de Albuquerque; Marin, Lauro Juliano; Aquino, Víctor Hugo; Figueiredo, Luíz Tadeu Moraes

doi:10.1590/s0036-46652002000300003

Cited by 15 publications

(17 citation statements)

References 17 publications

(27 reference statements)

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“…According to these authors, in the initial test, CMV genome was detected in 15.8% of samples (59 patients). Although the rate of positive samples was not stable throughout the above follow-up study, the difference in values of initially positive samples with the present study could be explained by the high prevalence of CMV infection in Brazil, about 95% 7,29 .…”

Section: Discussioncontrasting

confidence: 73%

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Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

Cunha

Aquino

Mariguela

et al. 2011

Rev. Inst. Med. trop. S. Paulo

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SUMMARYBackground: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. Methods: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 -cell count and opportunistic pathogens, including CMV. Semi nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. Results: In thirty patients (31%) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9%). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16%) followed by gB3 (35.48%). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm 3 (p = 0.0017), and almost all fatal cases (80%) had gB2 genotype. Conclusions: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

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Section: Discussioncontrasting

confidence: 73%

“…To amplify CMV DNA we used a nested-PCR technique previously reported by CUNHA et al 7 . The PCR was performed using CMV primers as follows: gB 1319 (5'-TGGAACTGGAACGTTTGGC3') and gB 1604 (5'GAAACGCGCGGCAATCGG-3') as external primers.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

Cunha

Aquino

Mariguela

et al. 2011

Rev. Inst. Med. trop. S. Paulo

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“…The reaction mixture of the PCR contained, in a total volume of 50 µL, 75 mM of Tris-HCl (pH 9), 2 mM of MgCl 2 , 50 mM of KCl, 20 mM of (NH 4 )2SO 4 , 50 µM of each one of the deoxynucleoside triphosphates, 0.3 µM of primers gB1 and gB2 (shown in Table 1) and 1 µg of DNA obtained from PBLs and enteric tissue. The reaction mixture was first incubated at 94 ºC for three min, the temperature was reduced to 80 ºC, and 1U of Taq DNA polymerase was added.…”

Section: Nested-pcrmentioning

confidence: 99%

“…The reaction mixture was first incubated at 94 ºC for three min, the temperature was reduced to 80 ºC, and 1U of Taq DNA polymerase was added. The PCR mixture was subjected to 30 cycles of 60 sec at 94 ºC, 60 sec at 55 ºC, 60 sec at 72 ºC, and finally to three min at 72 ºC 4 . Negative samples were subjected to a PCR for β-globin gene amplification, as a control for false negative results, confirming the integrity of DNA extracts and of the reaction mixture, as well as checking the efficacy of the PCR thermal cycles 7 .…”

Section: Nested-pcrmentioning

confidence: 99%

Cytomegalovirus in colorectal cancer and idiopathic ulcerative colitis

Mariguela

Chachá

Cunha

et al. 2008

Rev. Inst. Med. trop. S. Paulo

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SUMMARYCytomegalovirus (CMV) is a genus in the family Herpesviridae that has been associated with gastrointestinal syndromes. In this work we looked for a possible association of CMV infection with colorectal cancer and ulcerative colitis (UC). Blood and enteric tissue samples of 14 patients with colorectal cancer and of 21 with UC were subjected to a nested-PCR that amplifies part of the gB gene of CMV and also to immunohistochemistry using a specific monoclonal antibody to IE 76kDa protein of CMV. CMV was detected by nested-PCR in the blood and/or the enteric tissue of nine (64.3%) colorectal cancer and 16 (76.2%) ulcerative colitis patients. In the immunohistochemistry it was observed that 12 (12/21, 57.1%) positive enteric tissue samples of patients with UC and none from patients with colorectal cancer (0/14) were positive to CMV. The positivity of CMV infections in the UC patient group (12/21, 57.1%) showed by both techniques, was significantly higher (p = 0.015) than that observed for colorectal cancer patients (2/14, 14.3%). These results suggest an association of ulcerative colitis with CMV infection of the enteric tissue.

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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 …”

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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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Diagnosis of cytomegalovirus infections by qualitative and quantitative PCR in HIV infected patients

Cited by 15 publications

References 17 publications

Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

Cytomegalovirus in colorectal cancer and idiopathic ulcerative colitis

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

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