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Different cytomegalovirus glycoprotein B genotype distribution in serum and cerebrospinal fluid specimens determined by a novel multiplex nested PCR.

ABSTRACT: A novel and highly sensitive multiplex nested PCR assay has been developed for the simultaneous glycoprotein B (gB) typing of four gB genotypes of human cytomegalovirus (CMV) directly from clinical specimens. Specifically, a pair of primers to conserved regions of all gB genotypes within the gB gene (gB and gpUL55) was used for primary amplification. A mixture of nested primers to specific and conserved regions of each gB genotype was used for a secondary PCR amplification, yielding amplicons of different sizes for each gB genotype that could easily be differentiated by agarose gel electrophoresis. A total of 40 of 44 serum specimens and 26 of 26 cerebrospinal fluid (CSF) samples, which had previously tested positive for CMV in 66 of 70 AIDS patients with different CMV disease conditions, were gB genotyped by this novel assay. Significant differences in glycoprotein B genotype distribution between serum and CSF specimens were found (P = 0.001). gB genotype 3 (gB3) and gB2 were the most prevalent genotypes in sera (42.5%) and CSF (38.5%), respectively. A different distribution was also observed when only patients with CMV retinitis were studied (P = 0.005), suggesting a gB2 neuron tropism. Neither CMV disease nor any particular clinical manifestation of CMV disease was associated with gB genotypes (P > 0.05). A high incidence of mixed infection with the gB1 and gB3 genotypes (27.5%) was detected in serum specimens, indicating that reinfection and reactivation are common traits in advanced AIDS patients.

SUBMITTER: Tarrago D

PROVIDER: S-EPMC165308 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Different cytomegalovirus glycoprotein B genotype distribution in serum and cerebrospinal fluid specimens determined by a novel multiplex nested PCR.

Tarragó David D Quereda Carmen C Tenorio Antonio A

Journal of clinical microbiology 20030701 7

A novel and highly sensitive multiplex nested PCR assay has been developed for the simultaneous glycoprotein B (gB) typing of four gB genotypes of human cytomegalovirus (CMV) directly from clinical specimens. Specifically, a pair of primers to conserved regions of all gB genotypes within the gB gene (gB and gpUL55) was used for primary amplification. A mixture of nested primers to specific and conserved regions of each gB genotype was used for a secondary PCR amplification, yielding amplicons of ...[more]

PMID: 12843015

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Project description:Gurltia paralysans is an angio-neurotropic metastrongyloid nematode that infects domestic and wild cats, invading the veins of the subarachnoid space of the spinal cord and mainly causing progressive paralysis of the pelvic limbs. The definitive diagnosis of feline gurltiosis can only be achieved by post-mortem examination that reveals the presence of the nematode in the spinal cord vein vasculature. An early diagnosis with conclusive results is required since laboratory and imaging findings are not sufficient. Therefore, the purpose of this study was to detect the presence of G. paralysans, via semi-nested PCR, in samples of cerebrospinal fluid (CSF) and the sera of domestic cats naturally infected with the parasite. A total of 12 cats with a diagnosis suggestive of feline gurltiosis were selected, and they underwent a complete neurological and imaging examination. DNA samples were analysed by semi-nested PCR, with universal (AaGp28Sa1/AaGp28Ss1) and specific (Gp28Sa3/Aa28Ss2) primers, for G. paralysans (G. paralysans 18S rRNA gene, partial sequence; ITS 1, 5.8S rRNA gene, and ITS 2, complete sequence; and 28S rRNA gene, partial sequence) and Aelurostrongylus abstrusus, obtaining amplifications of 356 and 300 bp, which indicated the presence or absence of nematode DNA, respectively. The presence of G. paralysans was detected in the CSF of four out of nine cats, and the sera of seven out of seven cats. In the sera analysis of five out of seven cats, a mixed infection with A. abstrusus was found, despite no alterations of the respiratory tract being observed during the necropsies. It is proposed that serum samples could be more effective than CSF in detecting the parasite by PCR analysis. Sequencing analysis showed high percentages of identity with G. paralysans, which indicated the feasibility of detection and the sensitivity/specificity of the method used, suggesting the implementation of semi-nested PCR as a routine diagnostic test for early and timely detection of feline gurltiosis.

Dataset Information

Different cytomegalovirus glycoprotein B genotype distribution in serum and cerebrospinal fluid specimens determined by a novel multiplex nested PCR.

Publications

Different cytomegalovirus glycoprotein B genotype distribution in serum and cerebrospinal fluid specimens determined by a novel multiplex nested PCR.

Similar Datasets

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