Relative Research Between Change In T Lymphocyte Subpopulation And Congenital Human Cytomegalovirus Infection In Neonates

Objective Cord blood specimens from pregnant women were randomly collected and were detected HCMV by many Series of laboratory methods. Analysis of Normal maternity newborn congenital HCMV infection. At the same time, the proportion of T lymphocyte subpopulation incluing CD₃⁺, CD₄⁺ and CD₈⁺ from the cord blood samples were detected by flow cytometry assay, and calculated the CD₄⁺∕CD₈⁺ratio, analysis of congenital HCMV infection leads to changes in cellular immunity status, and further tracking of clinical manifestations.so as to provide an effective means of immunotherapy and to determine clinical efficacy.Methods 36 blood specimens from cord blood of normal puerpernants were randomly collected Since June 2010 March 2011 gathering in Hefei Maternal and Child Health. and were detected by following methods:(1) Isolation HCMV virus: Take 2ml cord blood anticoagulant, separated white blood cells and inoculated into human embryonic lung fibroblasts (HELF) culture, and daily observe HCMV cytopathic effect (Cytopathic effect, CPE) in microscope.(2) The peripheral blood mononuclear cells(PBMCs) were separated from plasma, then total DNA was extracted from PBMCs of 36 cord blood specimens. HCMV-DNA was detected by nested PCR and fluorescent quantitative PCR. (3) The peripheral blood mononuclear cells(PBMCs) were separated from plasma and were detected by HCMV-pp65 antigenemia assay. (4) 0.5ml cord blood detected by flow cytometry in T lymphocyte subsets incluing CD₃⁺, CD₄⁺ and CD₈⁺percentage, and calculated the CD₄⁺?CD₈⁺ratio. (5) After nested PCR amplification of the HCMV-DNA positive samples were sequenced and compared with the gene sequence database to determine the HCMV gB genotypes.Results (1) Results of Virus isolation: 36 blood specimens from cord blood of normal puerpernants were randomly collected, and inoculated into human embryonic lung fibroblasts (HELF) culture on three generations of blind passages, we could found 5 specimens appears HCMV characteristic CPE, virus isolation was 13.9% (5 / 36). (2) The positive detectable rate of HCMV-pp65 antigenemia in cord blood leukocytes of pregnant women was 16.7%(6∕36), Consistent with the virus isolation rate was 97.2%, of which common positive rate was 83.3%. (3) Results of nested PCR assay: 36 blood specimens from cord blood of normal puerpernants were randomly collected. The peripheral blood mononuclear cells(PBMCs) were separated from plasma, then total DNA was extracted from PBMCs of 36 cord blood specimens. HCMV-DNA were amplified by nested PCR. 6μl the inner PCR product( with 2μl loading buffer ) was subjected to gel electrophoresis in 1.2% agarose gel, by agarose gel electrophoresis Specific bands can be seen at about 261bp under UV light. Seven specific bands were observed at about 261bp. The positive rate of HCMV gB DNA detected by nested PCR was 22.2%(7∕36). (4) Results of fluorescent quantitative PCR assay:36 blood specimens from cord blood of normal puerpernants were randomly collected. The peripheral blood mononuclear cells(PBMCs) were separated from plasma, then total DNA was extracted from PBMCs of 36 cord blood specimens. HCMV-DNA were amplified by fluorescent quantitative PCR. The positive rate of HCMV DNA detected by fluorescent quantitative PCR was 30.6%(11∕36) according to the positive criteria of kits. (5) Analysis the sequence of HCMV gB DNA Nested PCR amplification products:Positive samples by nested PCR is consistent with primer sequence designed by nested PCR and were compared with the gene sequence database. All of the positive blood samples were proved to be HCMV gB2 type. (6) Results of changes in T lymphocytes subsets by flow cytometry assay:The CD₃⁺lymphocyte percentage in the HCMV DNA positive group and the negative group by fluorescent quantitative PCR assay respectively was (73.11±8.88)% and (71.66±8.59)%, and there was no significant difference between two groups (Pï¹¥0.05). The CD₄⁺lymphocyte percentage in the HCMV DNA positive group and the negative group respectively was (47.52±8.53)% and (52.476±7.25)%, and there was no significant difference between two groups (Pï¹¥0.05).However, there was a downward trend of CD₄⁺lymphocyte percentage in the HCMV DNA positive group compared with the HCMV DNA negative group. The CD8+lymphocyte percentage in the HCMV DNA positive group and the negative group by fluorescent quantitative PCR assay respectively was (25.84±4.75)% and (18.07±4.06)%, and the difference between two groups was significant (P﹤0.01). The CD₄⁺∕CD₈⁺ratio of HCMV DNA positive group and the negative group respectively was 1.89±0.45 and 3.026±0.75, and the difference between two groups was significant (P﹤0.01). (7) Connection with Neonates clinical manifestations found that a case of congenital HCMV active infection showed intrauterine growth retardation newborns, the HCMV virus isolation, HCMV-pp65 antigenemia test, HCMV UL55 gene nested PCR detection and HCMV-DNA fluorescence quantitative PCR testing was positive, CD₃⁺, CD₄⁺ and CD₈⁺ lymphocyte percentage was 66.4%, 36.2% 29.5%, CD₄⁺?CD₈⁺ratio was 1.21.Conclusion (1) According to the results of virus isolation as the "gold standard" to determine the initial recognition of congenital HCMV active infection, HCMV active infection also could initial confirmed by sequencing of PCR detection of HCMV IE, UL83 gene and nested PCR detection of UL55 gene positive and HCMV-pp65 antigenemia positive. (2) Through the virus isolation, HCMV-pp65 antigenemia test, HCMV UL55 gene nested PCR detection and quantitative HCMV-DNA PCR, in 36 blood specimens the normal newborn maternity HCMV congenital infection rate was 30.6%, of which, HCMV congenital active infection was 16.7%. (3) The proportion of T lymphocyte subpopulation incluing CD₃⁺, CD₄⁺ and CD₈⁺percentage from 36 cord blood specimens were detected by flow cytometry assay, and calculated the CD₄⁺?CD₈⁺ratio. We found there was a downward trend of CD₄⁺lymphocyte percentage in the HCMV DNA positive group compared with the negative group by fluorescent quantitative PCR assay. The CD₈⁺lymphocyte percentage in the HCMV DNA positive group and the HCMV DNA negative group by fluorescent quantitative PCR assay respectively was (25.84±4.75)% and (18.07±4.06)%, and the difference between two groups was significant (P﹤0.01). The CD₄⁺?CD₈⁺ratio of them respectively was 1.89±0.45and 3.026±0.75, and the difference between two groups was significant (P﹤0.01). Thus may determined HCMV infection related to cells immune response change. (4) A case of newborn infants weighing only 2.2 kg at birth have been detected in36 cases , meet the diagnostic criteria for intrauterine growth retardation. We need to establish follow-up files as soon as possible, and regularly home medical examination in newborns with HCMV congenital active infection urgently.